Compositions and methods for advanced glycosylation endproduct-mediated modulation of amyloidosis

ABSTRACT

The present invention relates generally to the non-enzymatic glycosylation of amyloidogenic proteins and the consequent formation of advanced glycosylation endproducts (AGEs). It has been found that formation of AGE-amyloidogenic proteins can enhance amyloidosis. The invention further relates to compositions and methods for the prevention and treatment of amyloidosis associated with amyloid diseases, particularly neurodegenerative disease and Type II diabetes, and more particularly Alzheimer&#39;s disease. In a specific example, aggregation of an amyloidogenic peptide, βAP, is enhanced by the glycosylation reaction of βAP to form AGE-βAP as defined herein. Accordingly, the invention extends to a method for modulating the in vivo aggregation of amyloid polypeptides and associated amyloidosis by controlling the formation and presence of AGE-amyloid polypeptide. A corresponding diagnostic utility comprises the measurement of the course and extent of amyloidosis by a measurement of the presence and amount of AGEs and particularly, AGE-amyloid. An assay is included that may use the AGE-amyloid polypeptide of the present invention to identify disease states characterized by the presence of AGE-amyloid. Additionally, such an assay can be utilized to monitor therapy and thus adjust a dosage regimen for a given disease state characterized by the presence of AGE-amyloid.

RELATED APPLICATIONS

The present application is a Continuation-in-part of ApplicationPCT/US95/01380, filed Feb. 2, 1995, which is a Continuation-in-part ofU.S. application Ser. No. 08/311,768, filed Sep. 23, 1994, nowabandoned, which is a Continuation-in-part of co-pending U.S.application Ser. No. 08/457,169, filed Jun. 1, 1995, which is acontinuation of U.S. application Ser. No. 08/191,579, filed Feb. 3,1994, now abandoned, of which the instant Application claims the benefitof the filing date pursuant to 35 U.S.C. §§ 120 and 365, and which isspecifically incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates generally to the non-enzymaticglycosylation of amyloid proteins and the often consequent formation ofadvanced glycosylation endproducts (AGEs). Formation of AGE-amyloid canresult in disease conditions or complications. The inventionparticularly relates to compositions and methods for the prevention andtreatment of amyloidosis associated with neurodegenerative diseases, inparticular Alzheimer's disease, and amyloidosis associated with Type II(adult onset) diabetes.

BACKGROUND OF THE INVENTION Amyloidosis and the B-Amyloid Peptide

Amyloidosis generally refers to a physiological condition which involvesdeposition of insoluble polypeptides, termed amyloid polypeptides oramyloid proteins. There are a wide range of amyloid proteins found invarious tissues throughout a subject, and a number of pathologicalconditions associated with various amyloidoses. For example, multiplemyeloma can result in amyloidosis with the immunoglobulin proteins.Idiopathic familial Mediterranean fever also involves systemicamyloidosis. Perhaps the best known disease associated with amyloidosisis Alzheimer's disease.

Alzheimer's disease (AD) affects more than 30% of humans over 80 yearsof age, and as such, represents one of the most important healthproblems of developed countries (Evans et al., 1989, JAMA 262:2551-56;Katzman and Saitoh, 1991, FASEB J. 280:278-286). The etiology andpathogenesis of this progressive dementia is poorly understood, butsymptomatic disease is associated with deposits of amyloid plaques,cerebrovascular amyloid and neurofibrillary tangles in the brain andcerebrovasculature. The number of plaques in AD patients' brains aretypically 5- to 10 fold greater than in age-matched healthy controls.Increased levels of plaques may result from increased rate of synthesisof the components of the plaques, decreased rate of degradation, or somecombination of the two.

The primary protein component of plaques is the 42 amino acid (4.2 kDa)beta-Amyloid Peptide (βAP), which derives from a family of largerAmyloid Peptide Precursor (APP) proteins (Glenner and Wong, 1984,Biochem. Biophys. Res. Commun. 120:885-890; Glenner and Wong, 1984,Biochem. Biophys. Res. Commun. 122:1131-35; Goldgaber et al., 1987,Science 235:8778-8780; Kang et al., 1987, Nature 325:733-736; Robakis etal., 1987, Proc. Natl. Acad. Sci. USA 84:4190-4194; Tanzi et al., 1987,Science 235:880-884). The process of amyloidosis is poorly understood,but requires at least βAP. Recent evidence shows that βAP may be foundin extracellular spaces like cerebrospinal fluid (CSF) of the brain andconditioned media of many cell types. Since increased amount& of amyloiddeposits are present in AD brains, one simple hypothesis is thatincreased βAP production leads to increased amyloidosis. Messenger RNAsencoding the APP precursors of βAP increase about 2-fold in AD brains,which has suggested to some a possible 2-fold increase in rates oftranslation, which may explain increased amyloid plaque formation (e.g.,Jacobsen et al., 1991, Neurobiol. Aging 12:585-592, and references citedtherein; Palmert et al., 1989, Prog. Clin. Biol. Res. 317:971-984;Tanaka et al., 1990, Rinsho Byori 38:489-493; Tanaka et al., 1989,Biochem. Biophys. Res. Commun. 165:1406-1414). An example of anincreased efficiency of βAP production that correlates with increasedplaque levels is found in a rare genetically linked familial form ofAlzheimer's disease (Cai et al., 1992, Science 259:514-516; Citron etal., 1992, Nature 360:672-674; Mullan et al., 1992, Nature Genet.1:345-347), known as a Swedish disease involving a doublelysine-methionine (KM) to asparagine-leucine (NL) mutation in APP nearthe amino-terminus of βAP. This mutation increases the release ofextracellular βAP in cultured cells. However, while this observation maypartly explain amyloidosis in the Swedish disease (and Down's Syndrome),βAP peptide levels in CSF of AD and healthy patients are the same(Oosawa et al., 1993, Soc. Neurosci. Abst. 19:1038; Shoji et al., 1992,Science 258:126-129). Thus, although healthy subjects appear to possesssimilar quantities of βAP as AD patients, they nevertheless fail toaccumulate the high number and amount of amyloid plaques seen in theirAD counterparts.

Post-translational events may contribute to amyloidosis. Beyondincreased rates of translation, physiological events such as greaterefficiency of βAP production from its precursor, aggregation intofibrillar structures, and resistance to proteolysis may unbalancedegradative processes, resulting in plaque formation.

Aggregation of the components of amyloid is a critical step in thedevelopment of amyloidosis. Once formed, fibrillar aggregates of βAP areextremely stable and not easily degraded. Amyloid plaques may bepurified by their resistance to solubilization in boiling SDS anddigestion with a variety of proteases. Additional treatment with 80%formic acid or 6M guanidine thiocyanate eventually solubilizes a portionof the plaque material. The solubilized protein is primarily the 42amino acid βAP. Yet even after these harsh denaturation treatments,dimers, tetramers and large molecular weight aggregates containingimmunoreactive βAP are found. This resistance to solubilization intosoluble or monomeric components suggests extensive proteinmodifications.

Further experiments have shown that primary neuronal cultures treatedwith full length βAP 1-42 in soluble form remain viable. Thus, solubleβAP 1-42 shows no toxicity. In contrast, cultures treated with insolubleaggregates of βAP 1-42 show a toxic response (Pike et al., 1991, Eur. J.Pharm. 207:367-368; Pike et al., 1993, J. Neurosci. 13:1676-87). Thisexperiment suggests that the toxicity of βAP is related to its state ofaggregation. Thus, an understanding of the mechanism forming fibrilsand/or insoluble aggregates from soluble βAP may be critical topreventing toxicity and resulting neurodegenerative disease.

In the absence of increased soluble βAP in most cases of AD, thequestion remains how amyloid accumulates to a greater degree atdifferent rates. Synthetic βAPs corresponding to the first 28, 40, or 42amino acids of βAP (i.e., βAP 1-28, βAP 1-40 and βAP 142, respectively)display concentration-dependent aggregation kinetics in in vitroincubations. Fibrillar aggregates form in vitro and these appear similarto brain β-amyloid fibrils at the morphological level using electronmicroscopy and at the light microscopy and spectroscopic levels usingCongo Red and Thioflavin stains.

The more rapid kinetics of aggregation observed at μM concentrations ofsoluble βAP in vitro are only of limited relevance for insight into themechanism of fibril formation in vivo. At lower βAP concentrations, forinstance in the physiological range of about 5 nM, there is aconsiderable lag period before measurable aggregate is formed in vitro.This observation suggests that the rate limiting step in aggregationcould be formation of a “nucleus” or “seed” upon which additional βAPcan rapidly accumulate.

Amylin, Pancreatic Islet Cells, and Type II Diabetes

There are two broad types of diabetes: Type I (childhood onsetdiabetes), which is associated with destruction of the pancreatic betacells and loss of insulin, and other hormones, produced by these cells,and is treated with insulin; and Type II (adult onset diabetes), whichis associated with insulin resistance. Type II diabetics can be furtherdivided into Type IIA, characterized by high blood pressure, obesity andinsulin resistance, and Type IIB, which includes lean individuals, obeseinsulin sensitive individuals, and young individuals. Perhaps the mostsignificant distinction between Type I and Type II diabetes is theabsence of autoimmune disease in Type II diabetes; otherwise, thissyndrome is characterized by a similarly diverse array of symptoms andcauses.

One common characteristic of Type II diabetics is the presence ofamyloid plaques in the pancreas. Such plaques are found in 90% of TypeII diabetics upon autopsy. As with Alzheimer's disease, the presence ofamyloid plaques in the affected organ cannot be conclusivelydemonstrated until autopsy (see, Edgington, 1994, Bio/Technology12:591). Two groups independently identified the major component ofpancreatic amyloid plaques as a 37 amino acid polypeptide termed isletamyloid polypeptide (IAPP) (Westermark et al., 1987, Proc. Natl. Acad.Sci. USA 84:3881-85; Westermark et al., 1987, Am. J. Physiol.127:414-417), or amylin (Cooper et al., 1987, Proc. Natl. Acad. Sci. USA84:8628-32; Cooper et al., 1988, Proc. Natl. Acad. Sci. USA 85:7763-66);the peptides identified by both groups appear to be interchangeable(Amiel, 1993, Lancet 341:1249-50). In its soluble form, amylinantagonizes insulin, and thus appears to have a role in the regulationof bloodstream glucose levels (see, Edgington, supra).

However, at high concentration, amylin, like βAP, aggregates in aβ-pleated sheet structure, and forms fibrils that appear to be toxic(Lorenzo et al., 1994, Nature 368:756-760). In particular, this paperreports that human amylin fibrils are toxic to insulin-producing β-cellsof the adult pancreas of rats and humans. Amylin fibrils appear toinduce islet cell apoptosis, leading to cell dysfunction and death inType II diabetes mellitus (Lorenzo et al., supra).

Advanced Glycosylation Endproducts (AGEs)

The reaction between glucose and proteins has been known for some time.Its earliest manifestation was in the appearance of brown pigmentsduring the cooking of food. In 1912, Maillard observed that glucose orother reducing sugars react with amino acids to form adducts thatundergo a series of dehydrations and rearrangements to form stable brownpigments (Maillard, 1912, C.R. Acad. Sci. 154:66-68).

In the years that followed the initial discovery by Maillard, foodchemists studied the hypothesized reaction in detail and determined thatstored and heat-treated foods undergo nonenzymatic browning as a resultof the reaction between glucose and polypeptide chains, and that theproteins thereby become crosslinked and exhibitdecreased.bio-availability. At this point, it was determined that thepigments responsible for the development of the brown color as a resultof protein glycosylation possessed characteristic spectra andfluorescent properties; however, the chemical structure of the pigmentshad not been specifically elucidated.

The reaction between reducing sugars and food proteins discussed abovewas found in recent years to have its parallel in vivo. Thus, thenonenzymatic reaction between glucose and the free amino groups onproteins to form a stable amino, 1-deoxy ketosyl adduct, known as theAmadori product, has been shown to occur with hemoglobin, wherein arearrangement of the amino terminal of the B-chain of hemoglobin byreaction with glucose forms the adduct known as hemoglobin A_(1c). Thereaction has also been found to occur with a variety of other bodyproteins, such as lens crystallin, collagen and nerve proteins (see Bunnet al., 1975, Biochem. Biophys. Res. Commun. 67:103-109; Koenig et al.,1975, J. Biol. Chem. 252:2992-2997; Monnier and Cerami, in MaillardReaction in Food and Nutrition, ed. Waller, G. A., American ChemicalSociety 1983, pp. 431-448; and Monnier and Cerami, 1982, Clinics inEndocrinology and Metabolism 11:431-452).

Moreover, brown pigments with spectral and fluorescent propertiessimilar to those of late-stage Maillard products have also been observedin vivo in association with several long-lived proteins, such as lensproteins and collagen from aged individuals. An age-related linearincrease in pigment was observed in human dura collagen between the agesof 20 to 90 years (see Monnier and Cerami, 1981, Science 211:491-493;Monnier and Cerami, 1983, Biochem. Biophys. Acta 760:97-103; and Monnieret al., 1984, “Accelerated Age-Related Browning of Human Collagen inDiabetes Mellitus”, Proc. Natl. Acad. Sci. USA 81:583-587).Interestingly, the aging of collagen can be mimicked in vitro in a muchshorter period of time by crosslinking induced by incubation in solutionwith relatively high concentrations of glucose. The capture of otherproteins and the formation of adducts by collagen, also noted, istheorized to occur by a crosslinking reaction, and is believed to.account, for instance, for the observed accumulation of albumin andantibodies in kidney basement membrane (see Brownlee et al., 1983, J.Exp. Med. 158:1739-1744; and Kohn et al., 1984, Diabetes 33:57-59).

Glucose and other reducing sugars attach non-enzymatically to the aminogroups of proteins in a concentration-dependent manner. Over time, theseinitial Amadori adducts can undergo further rearrangements, dehydrationsand cross-linking with other proteins to accumulate as a complex familyof structures referred to as Advanced Glycosylation Endproducts (AGEs).Substantial progress has been made toward the elucidation of the roleand clinical significance of advanced glycosylation endproducts, so thatit is now acknowledged that many of the conditions heretofore attributedto the aging process or to the pathological effects of diseases such asdiabetes, are attributable at least in part to the formation of AGEs invivo.

As noted above, advanced glycosylation end products tend to accumulateon molecules with long half-lives, especially under conditions ofrelatively high sugar concentration. Thus, AGE accumulation can beindicative of protein half-life, sugar concentration, or both. Thesefactors have important consequences. Numerous studies have suggestedthat AGEs play an important role in the structural and functionalalteration which occurs during aging and in chronic disease.Additionally, advanced glycosylation endproducts are noted to accumulateto a greater extent in diabetic and other diseased tissue than in normaltissue.

The “family” of AGEs includes species which can be isolated andcharacterized by chemical structure, some being quite stable, whileothers are unstable or reactive. The reaction between reducing sugarsand the reactive groups of proteins may initiate the advancedglycosylation process. This process typically begins with a reversiblereaction between the reducing sugar and the reactive group to form aSchiff base, which proceeds to form a covalently-bonded Amadorirearrangement product. Once formed, the Amadori product undergoesfurther rearrangement to produce the AGE-modified compound.

In U.S. Pat. No. 4,665,192, a fluorescent chromophore was isolated andidentified that was found to be present in certain browned polypeptides,such as bovine serum albumin and poly-L-lysine, and was assigned thestructure 2-(2-furoyl)-4(5)-2(furanyl)-1H-imidazole. More recently,other advanced glycosylation products have been identified, e.g., asdescribed in Farmar et al., U.S. Pat. No. 5,140,048, issued Aug. 18,1992; pyrraline (Hayase et al., 1989, “Aging of Proteins: ImmunologicalDetection of a Glucose-derived Pyrrole Formed during Maillard Reactionin Vivo”, J. Biol. Chem. 263:3758-3764); and pentosidine (Sell andMonnier, 1989, “Structure Elucidation of a Senescence Cross-link fromHuman Extracellular Matrix”, J. Biol. Chem. 264:21597-602).

Based on their knowledge of the role of AGEs in disease, the presentinventors have sought to identify factors that enhance aggregation ofβAP, and more importantly to identify agents and methods to inhibit theaction of such factors and thus prevent amyloidosis, e.g., inAlzheimer's disease and other amyloid diseases. More particularly, theinvention seeks to discover the relationship between advancedglycosylation endproduct formation and amyloidosis. Prior to the instantinvention, there has been no appreciation of a relationship betweenamyloidosis and advance glycosylation endproduct formation.

The citation of references herein shall not be construed as an admissionthat such is prior art to the present invention.

SUMMARY OF THE INVENTION

The present invention is broadly directed to the discoveries about thenature of AGE modification of amyloidogenic polypeptides, and theconsequences of such modification for the pathology and therapeutictreatment of diseases or disorders associated with amyloidosis.

In particular, the inventors have discovered that AGE-amyloidpolypeptides, in particular AGE-β amyloid peptide (βAP), facilitatefurther aggregation of amyloid polypeptides, whether such amyloidpolypeptides are AGE-modified or not.

The inventors have further related this discovery to the enhancedability of AGE-amylin polypeptides to facilitate aggregation of amylin(whether AGE-modified or not), resulting in amyloidosis of pancreatictissue and death of pancreatic islet cells.

Thus, the invention relates to a method of modulating AGE-amyloidpolypeptide-mediated amyloidosis in a mammal by controlling theformation of AGE-amyloid polypeptides. In a specific aspect of theinvention, aggregation of βAP and amylin have been determined to beenhanced by the glycosylation reaction of βAP or amylin to form AGE-βAPor AGE-amylin as defined herein. Accordingly, the invention particularlyextends to a method for modulating the in vivo aggregation of βAP andassociated neurodegenerative amyloidosis by controlling the formationand presence of AGE-βAP. The invention further particularly extends to amethod for modulating the in vivo aggregation of amylin and associatedpancreatic islet cell amyloidosis by controlling formation and presenceof AGE-amylin.

It has also been discovered that individuals suffering from anamyloidogenic disease have more AGEs associated with the amyloidpolypeptides that form the amyloid plaques characteristic of thedisease. The presence and level of AGE-amyloid polypeptides may reflectthe total body burden of amyloid polypeptides and their age. Inparticular, patients with Alzheimer's disease have more AGEs associatedwith βAP than normal individuals of the same age, and patients with TypeII diabetes may have more AGEs associated with amylin than normalindividuals. Since the absolute levels of βAP in AD and normalindividuals is about the same, the presence of AGE-βAP can be indicativeor predictive of AD.

A corresponding diagnostic utility comprises the measurement of thecourse and extent of amyloidosis by a measurement of the presence andamount of AGE-amyloid polypeptides, and particularly AGE-βAP andAGE-amylin, as defined herein. An assay is included that may use theAGE-amyloid polypeptide of the present invention to identify diseasestates characterized by the presence of the AGE-amyloid polypeptide.Additionally, such an assay can be utilized to monitor therapy and thusadjust a dosage regimen for a given disease state characterized by thepresence of the AGE-amyloid polypeptide. In specific embodiments, thediagnostic assays of the invention may be used to monitor the presenceor level of AGE-βAP or AGE-amylin.

As noted above, AGE-amyloid polypeptide is useful as a marker of avariety of conditions in which the fluctuation in amyloid polypeptidelevels may reflect the presence or onset of dysfunction or pathology.Moreover, AGE-amyloid polypeptide is useful alone and in conjunctionwith known carriers and delivery vehicles; such as liposomes, for thetransport of therapeutic and other agents, including in certaininstances the AGE moieties themselves, across membranes and epitheliallayers, for example, and particularly the blood brain barrier, toparticular sites in a patient for treatment. The particular site ofinterest may be an amyloid plaque that recognizes an AGE-amyloidpolypeptide, such as the AGE-βAP, or AGE-amylin, or a portion thereof.

The presence of high levels of AGE-amyloid polypeptides in amyloidogenicdiseases indicates that the normal clearance mechanisms for suchpolypeptides are faulty. Therefore, in a further aspect, the presentinvention provides compositions and methods for stimulating or inducingmechanisms of recognition and removal of AGE-amyloid in an animal, i.e.,the invention contemplates activation of the scavenger system in ananimal's body to remove the amyloid plaques. Such scavenger systemsinclude the activity of phagocytic cells, e.g., macrophages and, inneural tissue, microglial cells.

Accordingly, the invention provides for stimulating or activating thenatural scavenger systems by administration of stimulatory agents,including but not limited to, an advanced glycosylation endproduct, anAGE bound to a carrier, the fluorescent chromophore2-(2-furoyl)-4(5)-(2-furanyl)-1H-imidazole (FFI) bound to a carrier, amonokine (e.g., lymphokine or cytokine) that stimulates phagocytic cellsin the animal to increase the activity of recognizing and removingAGE-amyloid, and mixtures thereof. In a specific aspect, the AGE is anAGE-amyloid polypeptide.

Accordingly, the invention provides a method of preparing AGE-amyloidpolypeptide, in particular AGE-βAP or AGE-amylin, which comprisesincubation with an advanced glycosylation endproduct or a compound whichforms advanced glycosylation endproducts for a length of time sufficientto form said AGE-amyloid polypeptide, e.g., AGE-βAP or AGE-amylin.

Pharmaceutical compositions are also disclosed that comprise anAGE-amyloid polypeptide in combination with a pharmaceuticallyacceptable carrier. Such pharmaceutical compositions may include anadditional active agent(s) in some instances, and may be prepared andused for oral, parenteral or topical, e.g., transdermal, sublingual,buccal or transmucosal delivery. As stated, the pharmaceuticalcompositions can be in the form of a liposome in certain instances.

Generally, the therapeutic methods of the present invention contemplatethe inhibition of in vivo amyloid aggregation by the administration ofan agent or a pharmaceutical composition containing such agent or aplurality of such agents, for the inhibition of the formation ofadvanced glycosylation endproducts involving any or all of the amyloidpolypeptide and amyloid precursor polypeptide, and materials subject tosuch in vivo aggregation. Such agents comprise antagonists of advancedglycosylation, and include antibodies to AGEs, antibodies to AGE-amyloidpolypeptide, in particular AGE-βAP and AGE-amylin, as well as otherligands that would bind and neutralize the foregoing antigens. Suitableagents may also be selected from those agents that are reactive with anactive carbonyl moiety on an early glycosylation product, and preferablyare selected from aminoguanidine, a-hydrazinohistidine, analogs ofaminoguanidine, and pharmaceutical compositions containing any of theforegoing, all as recited in detail herein. The invention set forthherein contemplates the discovery of additional agents that may then beused in like fashion and for like purpose.

Accordingly, it is a principal object of the present invention tomodulate and control the in vivo aggregation of amyloid polypeptidesleading to amyloidosis by controlling the formation of advancedglycosylation endproducts (AGEs), and particularly AGEs involving suchamyloid polypeptides.

It is a further object of the present invention to provide a method forthe prognosis, monitoring, and/or diagnosis of conditions in whichabnormal amyloid accumulation is a characteristic, by detecting andmeasuring the presence and extent of AGE-amyloid polypeptide formation.

It is a still further object of the present invention to provide amethod for diagnosing and treating diseases associated with amyloidosis.It is a particular object of the invention to provide a method fordiagnosing, monitoring, and treating neurodegenerative diseasesassociated with amyloidosis, in particular Alzheimer's disease, bymeasuring and inhibiting the formation of AGE-βAP. It is anotherparticular object to provide a method for diagnosing, monitoring, andtreating diabetes Type II by measuring and inhibiting the formation ofAGE-amylin.

It is a still further object of the present invention to provide amethod for identifying new drugs and corresponding agents capable oftreating abnormal amyloid polypeptide aggregation, in one aspect by useof an assay involving AGE-amyloid polypeptide, in particular AGE-βAP orAGE-amylin.

Still another object of the invention is to provide for removing amyloidplaques that have formed in a subject by activating the mechanisms forrecognition and removal of AGE-amyloid in the body of a subject, andwhich may be directly or indirectly responsible for a pathology.

It is yet another object to utilize AGE-amyloid polypeptides,particularly AGE-βAP and AGE-amylin, to treat systemic orneurodegenerative diseases associated with amyloidosis, in particularAlzheimer's disease and Type II diabetes, respectively.

It is still a further object of the present invention to identifyAGE-amyloid proteins and methods of inhibiting their formation ininstances or disease conditions where the presence or biologicalactivity of these AGE-amyloid proteins is detrimental to the hostorganism, or indicative of the presence of a disease state in the hostorganism.

Other objects and advantages will be apparent from a consideration ofthe ensuing detailed description which proceeds with reference to thefollowing illustrative drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 presents a graph demonstrating that fluorescent aggregates formedwith synthetic βAP depend on initial concentration of solublepolypeptide and time. Synthetic βAP 1-28 was incubated with 0.1 M sodiumacetate, pH 7.2. Aliquots were removed at different times formeasurement of fluorescence in the presence of Thioflavin-T. The mean of3 measurements ±standard error is plotted for 600 μM (diamonds), 300 μM(circles) and 150 μM (squares) concentrations of βAP.

FIG. 2 presents a graph demonstrating that fluorescent aggregateformation displays nucleation-dependent kinetics. Stable aggregates ofsynthetic βAP were formed in the presence or absence of glucose togenerate “AGE-βAP seed” and “βAP seed.” Seeds were added to 300 μMsoluble βAP and incubated for different times. The mean ±standard errorof triplicate Thioflavin-T fluorescence measurements is plotted as afunction of time for “AGE-βAP seed”+βAP (circles) and “βAP seed”+βAP(diamonds). Control incubations included “AGE-βAP seed” alone(triangles), “βAP seed” alone (squares) and soluble βAP without seeding(crosses).

FIG. 3 demonstrates that “AGE-βAP seeds” nucleate more aggregation atphysiological concentrations of βAP than “βAP seeds.” 10 nM ¹²⁵I-βAP1-40 was mixed with no seed (squares), “βAP seed” (diamonds) or “AGE-βAPseed” (circles) and incubated for the indicated times at 37° C. The mean±standard error of quadruplicate measurements is plotted.

FIG. 4 is a graph showing that glucose modifies the kinetics offluorescent aggregate formation. Soluble βAP (400 μM βAP 1-28), 0.1 Msodium acetate in 0.1 M phosphate buffer, pH 7.0 (squares), were mixedwith 0.1 M glucose (diamonds), 0.05 M aminoguanidine (AG, triangles), orglucose and aminoguanidine (circles). The mean ±standard error oftriplicate Thioflavin-T fluorescence measurements is plotted on thisgraph.

FIG. 5 presents data showing that amyloid plaque-enriched fractions ofAlzheimer's diseased pre-frontal cortex contain more AGE adducts per mgprotein than equivalently prepared fractions of age-matched,non-demented controls. Each control patient is represented by a circleand AD patients by triangles. Each symbol represents the average of atleast 4 independent measurements of immunoreactive AGE adducts for eachpatient sample with the mean of each patient group marked by a crosssymbol.

FIG. 6 presents photographs that demonstrate the co-localization of AGEand prion protein (PrP) in PrP associated lesions, which contain amyloiddeposits, characteristic of the spongiform encephalopathy found in theneurodegenerative disease scrapie. Brain tissue sections were obtainedfrom 300 day old hamsters intracranially infected with a strain ofhamster scrapie, reacted with control or specific polyclonal rabbitantisera, followed by a second alkaline phosphatase-conjugatedanti-rabbit antibody to detect rabbit antibodies. (A) Rabbit anti-RNaseat 1:500 dilution (control); (B) Rabbit anti-PrP at 1:500 dilution; (C)rabbit anti-AGE-RNase (Makita et al., 1992, J. Biol. Chem. 267:5133-38)at a 1:500 dilution. Note that similar structures are decorated by therabbit antisera in (B) and (C).

FIG. 7 presents absorption spectral data relating to association of aThioflavin-T-Amadori product conjugate with fibrillar β-amyloid peptidein vitro. (A) Absorption spectrum of Thioflavin from 200 to 650 nm afterpelleting of fibrillar β-amyloid peptide. (B) Absorption spectrum ofThioflavin-T before pelleting. (C) Absorption spectrum ofdithionitrobenzene after pelleting. (D) Absorption spectrum ofdithionitrobenzene before pelleting.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to the prevention and treatment of adegenerative systemic, local, or neurological disease associated withamyloidosis.

In one aspect, the invention provides compositions and methods toprevent the formation or cross-linking of AGE-modified proteins involvedin amyloidosis. In a particular embodiment, the invention relates to theprevention of amyloidosis of the β-amyloid peptide (βAP) by inhibitingthe formation of advanced glycosylation endproduct (AGE)-modified βAP.βAP is a component of the amyloid plaques associated with Alzheimer'sdisease (AD), as well as other amyloidogenic degenerative neurologicaldiseases. In another embodiment, the invention relates to the preventionof arnyloidosis of amylin by inhibiting the formation of advancedglycosylation endproduct (AGE)-modified amylin. Amylin is a component ofthe amyloid fibrils found with pancreatic islet cells in associationwith Type II diabetes. In other embodiments, the invention relates toAGE-modulated amyloidosis of immunoglobulins produced by multiplemyeloma, amyloidosis associated with serum amyloid A peptide, andamyloidosis of the protein associated with one of the variousspongiformn encephalopathies, i.e., prion protein (PrP) orscrapie-associated fibril (SAF) protein.

In another aspect, the invention provides for clearance of amyloidplaques by activating resident phagocytic cells that express AGEreceptors, increasing the AGE content of the plaque, or both.

The invention is based, in part, on the discovery that the level of AGEfound in brain samples from AD patients is significantly greater than insimilarly prepared samples from age-matched control subjects. Additionalevidence forming a basis, in part, for the invention is the observationthat AGE epitopes are located in amyloid plaques in hamster-adaptedmurine scrapie, a form of spongiformn encephalopathy. The invention isfurther based partially on experiments demonstrating thatAGE-modification of βAP enhances the efficiency of βAP aggregation, andthat an inhibitor of AGE formation, in particular, aminoguanidine (AG),can inhibit the AGE-enhanced aggregation of 3AP.

Numerous abbreviations and terms are used herein to simplify theterminology used, and to facilitate a better understanding of theinvention.

The terms “amyloid,” “amyloid plaque,” and “amyloid fibril” refergenerally to insoluble proteinaceous substances with particular physicalcharacteristics independent of the composition of proteins or othermolecules that are found in the substance. Amyloid can be identified byits amorphous structure, eosinophilic staining, and homogeneousappearance. Protein or peptide components of amyloid are termed herein“amyloid polypeptides,” and include, but are not limited to, βAP;scrapie protein precursor or prion protein; immunoglobulin, including κor λ light or heavy chains, or fragments thereof, produced by myelomas;serum amyloid A; β₂-microglobulin; apoA1; gelsolin; cystatin C;(pro)calcitonin; atrial natururetic factor; islet amyloid polypeptide,also known as amylin (see, Westermark et al., 1987, Proc. Natl. Acad.Sci. USA 84:3881-85; Westermark et al., 1987, Am. J. Physiol.127:414-417; Cooper et al., 1987, Proc. Natl. Acad. Sci. USA 84:8628-32;Cooper et al., 1988, Proc. Natl. Acad. Sci. USA 85:7763-66; Amiel, 1993,Lancet 341:1249-50); and the like. It should be noted that human and catamylin are amyloidogenic peptides, and aggregate spontaneously in vitroto form insoluble fibrils, whereas rat amylin, which differs from humanamylin at six amino acid residues, is non-amyloidogenic and does notform fibrils (see, Lorenzo et al., 1994, Nature 368:756-760). In aspecific aspect, the term “amyloid” is used herein to refer tosubstances that contain βAP, scrapie protein, or amylin. “Amyloidosis”refers to the in vivo deposition or aggregation of proteins to formamyloid plaques or fibrils.

As used herein, the term “AGE-” refers to the compound which it modifiesas the reaction product of either an advanced glycosylation endproductor a compound which forms AGEs and the compound so modified, such as theβAP or amylin moiety. AGE-amyloid polypeptide can be formed in vitro orin vivo by reacting an amyloidogenic polypeptide or amyloid as definedherein with an AGE, such as AGE-βAP or AGE-amylin, or with a compoundsuch as a reducing sugar, e.g., glucose, until the peptide is modifiedto form the AGE-peptide.

The term “glycosylation” is used herein to refer to the non-enzymaticreaction of reducing sugars with a nucleophile, in particular an aminegroup, on an amyloid polypeptide, such as βAP, which leads to formationof AGEs. These processes are well known in the art, as described above.An alternative term for this process that has come more frequently intouse is “glycation.”

A molecule is “antigenic” when it is capable of specifically interactingwith an antigen recognition molecule of the immune system, such as animmunoglobulin (antibody) or T cell antigen receptor. An antigenicpeptide contains at least about 5, and preferably at least about 10,amino acids. An antigenic portion of a molecule can be that portion thatis immunodominant for antibody or T cell receptor recognition, or it canbe a portion used to generate an antibody to the molecule by conjugatingthe antigenic portion to a larger, immunogenic carrier molecule forimmunization. A molecule that is antigenic need not be itselfimmunogenic, i.e., capable of eliciting an immune response withoutcoupling to such a carrier.

A composition comprising “A” (where “A” is a single protein orpolypeptide, DNA molecule, vector, recombinant host cell, etc.) issubstantially free of “B” (where “B” comprises one or more contaminatingproteins or polypeptides, DNA molecules, vectors, etc.) when at leastabout 75% by weight of the proteins or polypeptides, DNA, vectors(depending on the category of species to which A and B belong) in thecomposition is “A”. Preferably, “A” comprises at least about 90% byweight of the A+B species in the composition, most preferably at leastabout 99% by weight. It is also preferred that a composition besubstantially free of contamination, and generally that suchcompositions contain only a single molecular weight species having theactivity or characteristic of the species of interest.

The phrase “pharmaceutically acceptable” refers to molecular entitiesand compositions that are physiologically tolerable and do not typicallyproduce an allergic or similar untoward reaction, such as gastric upset,dizziness and the like, when administered to a human. Preferably, asused herein, the term “pharmaceutically acceptable” means approved by aregulatory agency of the Federal or a state government or listed in theU.S. Pharmacopeia or other generally recognized pharmacopeia for use inanimals, and more particularly in humans. The term “carrier” in thiscontext refers to a diluent, adjuvant, excipient, or vehicle with whichthe compound is administered. Pharmaceutically acceptable carriers canbe sterile liquids, such as water and oils, including those ofpetroleum, animal, vegetable or synthetic origin, such as peanut oil,soybean oil, mineral oil, sesame oil and the like. Water or aqueoussaline solutions and aqueous dextrose and glycerol solutions arepreferably employed as carriers, particularly for injectable solutions.Suitable pharmaceutical carriers are described in “Remington'sPharmaceutical Sciences” by E. W. Martin.

The term “adjuvant” refers to a compound or mixture that enhances theimmune response to an antigen. An adjuvant can serve as a tissue depotthat slowly releases the antigen and also as a lymphoid system activatorthat non-specifically enhances the immune response (Hood et al.,Immunology, Second Ed., 1984, Benjamin/Cummings: Menlo Park, Calif., p.384). Often, a primary challenge with an antigen alone, in the absenceof an adjuvant, will fail to elicit a humoral or cellular immuneresponse. Adjuvants include, but are not limited to, complete Freund'sadjuvant, incomplete Freund's adjuvant, saponin, mineral gels such asaluminum hydroxide, surface active substances such as lysolecithin,pluronic polyols, polyanions, peptides, oil or hydrocarbon emulsions,keyhole limpet hemocyanins, and potentially useful human adjuvants suchas BCG (bacille Calmette-Guerin) and Corynebacterium parvum. Preferably,the adjuvant is pharmaceutically acceptable.

A disease or disorder is associated with amyloidosis when amyloiddeposits or amyloid plaques are found in or in proximity to tissuesaffected by the disease, or when the disease is characterized byoverproduction of a protein that is or can become insoluble. The amyloidplaques may provoke pathological effects directly or indirectly by knownor unknown mechanisms. Examples of amyloid diseases include, but are notlimited to, systemic diseases, such as chronic inflammatory illnesses,multiple myeloma, macroglobulinernia, familial amyloid polyneuropathy(Portuguese) and cardiomyopathy (Danish), systemic senile amyloidosis,familial amyloid polynephropathy (Iowa), familial amyloidosis (Finnish),Gerstmann-Straussler-Scheinker syndrome, familial amyloid nephropathywith urticaria and deafness (Muckle-Wells syndrome), medullary carcinomaof thyroid, isolated atrial amyloid, and hemodialysis-associatedamyloidosis (HAA); and neurodegenerative diseases.

Type II diabetes is associated with amyloid fibrils or deposits of thepancreas, in particular the islet cells that produce insulin. As withAlzheimer's disease amyloid plaques, the amyloid plaques or fibrilsprovoke pathological effects. In particular, concentrations of humanamylin at which fibrils form are toxic for human and rat pancreaticislet insulin-producing fl-cells (Lorenzo et al., 1994, Nature368:758-760). Accordingly, in a specific embodiment, the inventionrelates to Type II diabetic amyloidosis.

Chronic inflammatory illnesses, such as idiopathic familialMediterranean fever, Muckle-Wells syndrome, chronic malarial infection,and the like, can result in expression of serum amyloid A, an acutephase protein which may undergo further processing and form amyloiddeposits and plaques. For example, in the Third World, chronic malariacan lead to amyloidosis of the spleen and/or liver of an individual. Theresulting organ failure can ultimately lead to death. Multiple myelomais associated with overproduction of immunoglobulins, whichimmunoglobulins or fragments thereof can form amyloid deposits andplaques in organs or tissues in contact with the circulatory system.Deposition of transthyretin can result in familial amyloidpolyneuropathy (Portuguese), familial amyloid cardiomyopathy (Danish),or systemic senile amyloidosis. Hemodialysis-associated amyloidosis is acomplication among long-term hemodialysis patients, in whichβ₂-microglobulin is a major protein constituent of the amyloid fibrils(Drueke, 1991, Miner. Electroyte Metab. 17:261-272; Geyjo et al., 1985,Biochem. Biophys. Res. Commun. 129:701-706; Gorevic et al., 1986, Proc.Natl. Acad. Sci. USA 83:7908-12; Shirahama et al., 1985, Lab. Invest.53:705-709).

As noted above, in addition to systemic amyloidosis, the presentinvention relates particularly to neurodegenerative diseases involvingamyloidosis. The term “neurodegenerative disease” refers to a disease ordisorder of the nervous system, particularly involving the brain, thatmanifests with symptoms characteristic of brain or nerve dysfunction,e.g., short-term or long-term memory lapse or defects, dementia,cognition defects, balance and coordination problems, and emotional andbehavioral deficiencies. Such diseases are “associated with amyloidosis”when histopathological (biopsy) samples of brain tissue from subjectswho demonstrate such symptoms would reveal amyloid plaque formation. Asbiopsy samples from brain, especially human brain, are obtained withgreat difficulty from living subjects or might not be available at all,often the association of a symptom or symptoms of neurodegenerativedisease with amyloidosis is based on criteria other than the presence ofamyloid deposits, such as plaques or fibrils, in a biopsy sample.

In a specific embodiment, according to the present invention theneurodegenerative disease associated with amyloidosis is Alzheimer'sdisease (AD). In other embodiments, the disease may be the rare Swedishdisease characterized by a double KM to NL mutation in amyloid precursorprotein (APP) near the amino-terminus of the βAP portion of APP (Levy etal., 1990, Science 248:1124-26). Another such disease is hereditarycerebral hemorrhage with amyloidosis (HCHA or HCHWA)-Dutch type(Rozemuller et al., 1993, Am. J. Pathol. 142:1449-57; Roos et al., 1991,Ann. N.Y. Acad. Sci. 640:155-60; Timmers et al., 1990, Neurosci. Lett.118:223-6; Haan et al., 1990, Arch. Neurol. 47:965-7). Other suchdiseases known in the art and within the scope of the present inventioninclude, but are not limited to, sporadic cerebral amyloid angiopathy,hereditary cerebral amyloid angiopathy, Down's syndrome,Parkinson-dementia of Guam, and age-related asymptomatic amyloidangiopathy (see, e.g., Haan and Roos, 1990, Clin. Neurol. Neurosurg.92:305-310; Glenner and Murphy, 1989, N.

Neurol. Sci. 94:1-28; Frangione, 1989, Ann. Med. 21:69-72; Haan et al,1992, Clin. Neuro. Neurosurg. 94:317-8; Fraser et al., 1992, Biochem.31:10716-23; Coria et al., 1988, Lab. Invest. 58:454-8). The actualamino acid composition and size of the βAP involved in each of thesediseases may vary, as is known in the art (see above, and Wisniewski etal., 1991, Biochem. Biophys. Res. Commun. 179:1247-54 and 1991, Biochem.Biophys. Res. Commun. 180:1528 [published erratum]; Prelli et al., 1990,Biochem. Biophys. Res. Commun. 170:301-307; Levy et al., 1990, Science248:1124-26).

In a further aspect, the neurodegenerative disease is a subacutespongiform encephalopathy, such as but not limited to, scrapie,Creutzfeldt-Jakob disease, Gerstmann-Straussler disease, kuru, chronicwasting disease of mule-deer and elk, bovine spongiform encephalopathyof cattle, and mink transmissible encephalopathy.

The instant invention contemplates the treatment of animals, and morepreferably, mammals, including humans, as well as mammals such as dogs,cats, horses, cows, pigs, guinea pigs, mice and rats.

Treatment of Neurodegenerative Amyloidosis by Inhibiting AGE

In one aspect, the present invention provides for therapeutic treatmentfor the prevention or inhibition of amyloidosis associated with diseasesor disorders, e.g., neurodegenerative diseases, in particularAlzheimer's disease. In broad aspect, the therapeutic method of theinvention involves administration of an agent that is capable ofcontrolling the production, formation, or accumulation of advancedglycosylation endproducts. Such agents include, but are not limited to,antibodies against advanced glycosylation endproducts, ligands,including AGE receptors and active fragments thereof, capable of bindingto and neutralizing advanced glycosylation endproducts, and compoundscapable of inhibiting the formation of advanced glycosylationendproducts. In particular, the invention relates to an inhibitor ofglycosylation, preferably an inhibitor of AGE formation, to the brain ofa subject believed to be in need of such treatment. Such an agent istermed herein “capable of inhibiting the formation of AGEs”, oralternatively an “inhibitor of AGE formation”, “inhibitor of advancedglycosylation,” or an “agent that inhibits advanced glycosylation.”

The present invention further contemplates a dual therapeutic strategy,where agents that inhibit advanced glycosylation, such asaminoguanidine, may be administered to inhibit in vivo AGE-amyloidpolypeptide formation and consequent initiation of amyloid polypeptideaggregation, plaque formation, and amyloidosis; and to react with anybyproducts of an ongoing amyloid glycosylation to prevent reaction ofthese byproducts with proteins, particularly other amyloid polypeptides,resulting in proteolytically resistant cross-links in the amyloidplaque.

The therapeutic (and, as discussed below, the diagnostic) methods of thepresent invention contemplate the use of agents that have an impact onthe formation of AGE-amyloid. Among these agents, antibodies to AGEs andother ligands may be prepared and used.

The rationale of the invention is to use agents which block thepost-glycosylation step, i.e., the formation of fluorescent chromophoresand/or molecular crosslinks whose presence is associated with, and leadsto, the adverse sequelae of glycosylation. An ideal agent would preventthe formation of AGE-associated chromophores and/or cross-links bridgingproteins and covalently trapping proteins onto other proteins, such asoccurs in amyloid plaques.

The present invention does not attempt to prevent initial proteinglycosylation reactions, as it would be nearly impossible to use agentswhich prevent the reaction of glucose with protein amino groups. Theagents that are capable of preventing initial glycosylation are likelyto be highly toxic, and since the initial glycosylation comes toequilibrium in about three weeks, there is inadequate time available toachieve this objective. Instead, the ideal agent would prevent orinhibit the long-term, post-glycosylation steps that lead to theformation of the ultimate advanced glycosylation end products that are adirect cause of the pathology associated with amyloidosis.

In a further aspect of the invention, an inhibitor of the formation ofAGEs includes compounds that react with a carbonyl moiety of an earlyglycosylation product. Representative of such advanced glycosylationinhibitors are aminoguanidine, lysine and α-hydrazinohistidine. In aspecific embodiment, the inhibitor is aminoguanidine (AG) andderivatives thereof. Pharmaceutical compositions and methods involvingAG and derivatives thereof are well known, as described in U.S. Pat. No.4,758,583, issued Jul. 19, 1988; U.S. Pat. No. 4,908,446, issued Mar.13, 1990; U.S. Pat. No. 4,983,604, issued Jan. 8, 1991; U.S. Pat. No.5,100,919, issued Mar. 31, 1992; U.S. Pat. No. 5,106,877, issued Apr.21, 1992; U.S. Pat. No. 5,114,943, issued May 19, 1992; U.S. Pat. No.5,128,360, issued Jul. 7, 1992; U.S. Pat. No. 5,130,324, issued Jul. 14,1992; U.S. Pat. No. 5,130,337, issued Jul. 14, 1992; U.S. Pat. No.5,137,916, issued Aug. 11, 1992; U.S. Pat. No. 5,140,048, issued Aug.18, 1992; U.S. Pat. No. 5,175,192, issued Dec. 29, 1992; U.S. Pat. No.5,218,001, issued Jun. 8, 1993; U.S. Pat. No. 5,221,683, issued Jun. 22,1993; U.S. Pat. No. 5,238,963, issued Aug. 24, 1993; U.S. Pat. No.5,243,071, issued Sep. 7, 1993; and U.S. Pat. No. 5,254,593, issued Oct.19, 1993. Other inhibitors of AGE formation are described in U.S.applications Ser. No. 07/652,575, filed Feb. 8, 1991; Ser. No.07/889,141, filed May 27, 1992; Ser. No. 07/896,854, filed May 15, 1992;Ser. No. 07/986,661, filed Dec. 8, 1992; Ser. No. 07/986,662, filed Dec.8, 1992; Ser. No. 08/027,086, filed Mar. 5, 1993; and Ser. No.08/095,095, filed Jul. 20, 1993. Each of the foregoing patents andpatent applications is specifically incorporated herein by reference inits entirety. Such inhibitors of AGE formation can be administereddirectly to the brain or cerebrospinal fluid, e.g., by direct cranial orintraventricular injection, or may pass through the blood brain barrierfollowing administration by parenteral injection, oral administration,skin absorption, etc.

Accordingly, such compounds include a variety of hydrazine derivativeshaving, for example, a generic formula as follows:

wherein R is a group of the formula

and R₁ is hydrogen or a lower alkyl group of 1-6 carbon atoms, ahydroxyethyl group, or together with R₂ may be a lower alkylene bridgeof 2-4 carbon atoms; R₂ is hydrogen or a lower group alkyl of 1-6 carbonatoms or together with R₁ or R₃ is a lower alkylene bridge of 2-4 carbonatoms, amino, hydroxy, or an aminoalkylene group of the formula

wherein n is an integer of 2-7 and R₆ and R₇ are independently a loweralkyl group of 1-6 carbon atoms or together form a part of a cycloalkylor heterocyclic ring containing from 1 to 2 heteroatoms, of which atleast one is nitrogen; and the second of said heteroatoms is selectedfrom the group consisting of nitrogen, oxygen, and sulfur; with theproviso that when the second of said heteroatoms of the heterocyclicring is nitrogen and forms a piperazine ring; it may be optionallysubstituted by a substituent that is identical to the portion of thecompound on the first nitrogen of the piperazine ring; R₃ is hydrogen, alower alkyl group of 1-6 carbon atoms, or together with R₂ or R₄ is alower alkylene bridge of 2-4 carbon atoms; R₄ is hydrogen, a lower alkylgroup of 1-6 carbon atoms or together with R₃ is a lower alkylene bridgeof 2-4 carbon atoms; or an amino group; R₅ is hydrogen, or a lower alkylgroup of 1-6 carbon atoms; with the proviso that at least one of R₁, R₂,R₃, R₄ or R₅ is other than hydrogen; or R is an acyl or a loweralkylsulfonyl group of up to 10 carbon atoms and R₁ is hydrogen; andtheir pharmaceutically acceptable acid addition salts.

In a particular aspect, where the disease associated with amyloidosis isa neurodegenerative disease, preferably an inhibitor of AGE formationmay be capable of crossing the blood brain barrier. The blood brainbarrier of subjects suffering from brain amyloidosis is often found indeteriorated condition, and this facilitates the ability of agentsadministered parenterally to traverse the barrier. In anotherembodiment, the inhibitor of AGE formation can be conjugated with atargeting molecule, such as transferrin, for which there are receptorson the blood brain barrier. In a further embodiment, the inhibitor canbe modified to have decreased polarity, or increased hydrophobicity, asmore hydrophobic (less polar) agents cross the blood brain barrier morereadily. In a further embodiment, hydrophobic (non-polar) inhibitors ofAGE formation can be selected and used. In yet another embodiment, theinhibitor of advanced glycosylation can be administered in a liposome,particularly a liposome targeted to the blood brain barrier.Administration of pharmaceutical agents in liposomes is known (seeLanger, 1990, Science 249:1527-1533; Treat et al., in Liposomes in theTherapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler(eds.), Liss, New York, pp. 353-365 (1989); Lopez-Berestein, ibid., pp.317-327; see generally ibid.).

These and other strategies for directing therapeutic agents across theblood brain barrier are known in the art, and contemplated by thepresent invention.

In another embodiment, inhibitors of AGE can be antibodies. Antibodiescan bind to and inactivate or mediate clearance of AGE-modified amyloidpolypeptides. In one aspect of the invention, the antibody described inMakita et al. (1992, J. Biol. Chem. 267:5133-38) can be used. Theinvention further provides for generation of antibodies to AGE epitopesof AGE-amyloid polypeptides. Such antibodies can be prepared usingtechniques well known in the art. Preferably, the immunogen used toprepare the antibodies is an AGE-amyloid protein. In a specific aspect,AGE-βAP can be used. In another embodiment, AGE-amylin can be used.

The AGE-βAP or AGE-amylin may be used to produce antibody(ies) tothemselves. Such antibodies can be produced and isolated by standardmethods including the well known hybridoma techniques. Generally,antibodies can be produced by immunization of an animal with AGE-βAP orAGE-amylin, free or conjugated with a carrier protein, such as but notlimited to keyhole limpet hemocyanin (KLH) or BSA, preferably admixedwith an adjuvant as defined above.

The term “antibody” includes any immunoglobulin, including antibodiesand fragments thereof that binds a specific epitope, and such generaldefinition is intended to apply herein. The term therefore encompassespolyclonal, monoclonal and chimeric antibodies, the last mentioneddescribed in further detail in U.S. Pat. Nos. 4,816,397 and 4,816,567.Also, an “antibody combining site” is that structural portion of anantibody molecule comprised of heavy and light chain variable andhypervariable regions that specifically bind antigen.

Exemplary antibodies include antibody molecules such as intactimmunoglobulin molecules, substantially intact immunoglobulin moleculesand those portions of an immunoglobulin molecule that contain the activebinding site, including those portions known in the art as Fab, Fab′,F(ab′)₂ and F(v), which portions are preferred for use in therapeuticmethods associated herein.

Fab and F(ab′)₂ portions of antibody molecules are prepared by theproteolytic reaction of papain and pepsin, respectively, onsubstantially intact antibody molecules by methods that are well-known.See for example, U.S. Pat. No. 4,342,566 to Theofilopolous et al. Fab′antibody molecule portions are also well-known and are produced fromF(ab′)₂ portions by reduction of the disulfide bonds linking the twoheavy chain portions as with mercaptoethanol, and followed by alkylationof the resulting protein mercaptan with a reagent such as iodoacetamide.

The phrase “monoclonal antibody” in its various grammatical forms refersto an antibody having only one species of antibody combining sitecapable of immunoreacting with a particular antigen. A monoclonalantibody thus typically displays a single binding affinity for anyantigen with which it immunoreacts. An antibody may be.prepared having aplurality of antibody combining sites, each immunospecific for adifferent antigen, e.g., a bispecific (chimeric) antibody.

While intravenous injection is a very effective form of parenteraladministration, other modes can be employed, including but not limitedto intraventricular, intramuscular, intraperitoneal, intra-arteriolar,and subcutaneous injection as well as oral, nasal and topicaladministration. Intraventricular injection may be preferred fortreatment of a neurodegenerative disease. Intravenous injection may bepreferred for systemic amyloidosis, or for treating amyloidosisassociated with Type II diabetes.

Preferably, the treatment is effected prophylactically, to prevent theinitial formation of amyloid seed, which may facilitate aggregation ofnon-AGE modified amyloid polypeptide, such as soluble βAP or solubleamylin, as well as AGE-amyloid polypeptide. In a particular embodiment,an inhibitor of AGE formation that can traverse the blood brain barrier,as described above, can be administered to a subject believed to be atincreased risk for Alzheimer's disease or another neurodegenerativedisease that involves amyloidosis to inhibit the onset or progression ofthe disease at an early stage. In another embodiment, the inhibitor ofAGE formation can be injected intraperitoneally or intravenously in asubject believed to be at increase risk for Type II diabetes, e.g., aperson suffering from obesity or one of the other conditions associatedwith Type II diabetes. For example, subjects that have a geneticpredisposition to AD or Type II diabetes can be treatedprophylactically. The actual dosage and treatment regimen for suchprophylaxis can be readily determined by the ordinary skilled physician,taking into account the route of administration, age and weight of thepatient, and the particular disease state for which the patient isundergoing treatment, as well as the stage thereof, and, of course, anyside effects of the inhibitor, efficacy of the inhibitor, in accordancewith customary medical procedures and practices.

The invention further contemplates that the inhibitors of AGE-formationcan be administered in conjunction with other therapies for thetreatment of diseases that involve amyloidosis. For example, fortreatment of a neurodegenerative disease, in particular Alzheimer'sdisease, an inhibitor of AGE-formation can be administered inconjunction with a therapy designed to inhibit production of βAP, suchas those described by Gandy et al., U.S. Pat. No. 5,242,932, issued Sep.7, 1993; Wagner et al., International Patent Publication No. WO93/09233, published May 13, 1993; and Buxbaum et al., European PatentPublication No. 0457295 A2, published 21 November 1991. In anotherexample, for treatment of Type II diabetes, administration of aninhibitor of AGE-formation can be effected in conjunction withadministration of one or more of sulfonylureas (drugs to increase thelevel of insulin production), insulin, hypertension medication, andimposition of a diet and exercise regimen.

Treatment of Amyloidosis by Increasing AGE Clearance

In accordance with the present invention, a method and associated agentsare disclosed for the inhibition and treatment of amyloidosis in animalsby stimulating the bodies of such animals to increase their recognitionof and affinity for advanced glycosylation endproducts. In particular,phagocytic cells such as monocytes, macrophages and/or microglial cellsare treated with an agent capable of causing the phagocytic cells toincrease their activity of recognizing and removing AGE-modified amyloidplaques.

The agents of the present invention comprise one or more stimulatorcompounds in turn, comprising a natural or synthetic advancedglycosylation endproduct alone or bound to a carrier, said carrierincluding a material selected from carbohydrates, proteins, syntheticpolypeptides, lipids, bio-compatible natural and synthetic resins,antigens, and mixtures thereof. The stimulator compounds could includeother advanced glycosylation endproducts that may be prepared from thereaction between sugars and other macromolecules, and monokines whichstimulate phagocytic cells to increase their activity toward advancedglycosylation endproducts (see. U.S. Pat. No. 4,900,747, issued Feb. 13,1990 to Vlassara et al., which is incorporated herein by reference inits entirety).

Accordingly, the stimulator compound may comprise the compound FFI boundto a protein such as albumin. Alternately, the stimulator compound maycomprise a synthetically derived advanced glycosylation endproduct whichis prepared, for example, by the reaction of glucose orglucose-6-phosphate with albumin. This reaction product can be usedalone or with a carrier in the same fashion as the FFI-albumin complex.In a specific aspect, the stimulator compound is an AGE-amyloidpolypeptide.

A monokine that functions as a stimulator compound comprises the proteinknown as Tumor Necrosis Factor (TNF) discovered and isolated by one ofthe inventors herein and named “cachectin.” This material may beadministered alone or in conjunction with other stimulator compounds.

In addition, the stimulator compounds of the present invention may beadministered in conjunction with materials identified hereinafter as“co-stimulatory agents.” The co-administration of the stimulatorcompound with the co-stimulatory agents has been found to potentiate theactivity of the former. Suitable co-stimulatory agents include monokinessuch as Interleukin-1 (IL-1) and gamma-interferon.

A further alternative embodiment of the method of the present inventionand one which may be practiced independently or conjointly with theabove recited method, is the ex vivo treatment of the phagocytic cellsto expose them to the stimulator compounds. For example, a patient maybe given an extracorporeal blood treatment in which blood is divertedout of the body from the arterial and venous system and is directedthrough a device which contains stimulator compounds and/orco-stimulatory agents which are suitably positioned to come in contactwith the phagocytic cells within the blood. The stimulator compoundsand/or co-stimulatory agents may be immobilized or may be allowed toenter the flow of the body fluid.

In the instance where the method comprises the in vivo administration ofthe stimulator compound and/or stimulatory agents, such administrationmay be accomplished by known techniques, including oral techniques andparenteral techniques such as intradermal, subcutaneous, intravenous, orintraperitoneal injection, catheterization or other conventional means.The stimulator compounds or mixtures of them may be prepared in suitablepharmaceutical compositions for such administration.

In a specific embodiment, AGE-modified amyloid polypeptides are usefulfor activating tissue phagocytic cells, such as macrophages, which canin turn metabolize amyloid deposits or plaques. Such an amyloidpolypeptide may have an amino sequence identical to the native aminoacid sequence, or it may be modified to include one or more additionalsites for non-enzymatic glycation and AGE formation. For example, ahistidine, asparagine, or glutamine residue (i.e., a polar or cationicresidue) may be substituted with lysine.

As noted above, administration of an AGE-modified amyloid polypeptidecan activate phagocytic mechanisms in tissue phagocytic cells. Inaddition to phagocytosing amyloid, such phagocytic cells may alsosecrete enzymes that help degrade amyloid, and may recruit other cellsthat can assist in the removal of amyloid.

In a further particular aspect, the AGE-βAP or other AGE-neural amyloidpolypeptides of this invention can be utilized as stimulants ofactivation of neural phagocytic cells, in particular microglia, toactivate the microglia to effect removal of AGEs and/or AGE-modifiedpolypeptides, and thus, amyloid. Such phagocytic cells are capable ofrecognizing and removing abnormal macromolecules by means of receptorson their surfaces which recognize specific chemical structures and bindthem. Once the abnormal macromolecule is recognized in this way, thephagocytic cell may internalize the macromolecule and may then degradeit. In some instances, the phagocytic cell may in addition secreteenzymes and other factors to help degrade the molecule or particleextracellularly if it cannot be internalized or to induce other cells toparticipate in such degradation. After the amyloid is removed, normalfunction of the affected area may resume.

In another specific aspect, the invention contemplates administration ofAGE-amylin to activate the body's absorption mechanisms to removepancreatic amyloid plaques or fibrils associated with Type II diabetes.

The present invention contemplates that the phagocytic cells can beactivated by exposure to stimulator compounds that potentiate thecapability of these cells with respect to their recognition and affinityfor, and capability to degrade, advanced glycosylation end products. Inparticular, the exposure of these cells to certain stimulator compoundshas been found to increase the number of receptors developed on thesecells and to thereby increase the capacity and efficiency of these cellswith respect to the recognition and degradation of advancedglycosylation endproducts. Thus, in a specific aspect, the AGE-βAP orAGE-amylin of the present invention can function as a stimulatorcompound, as can other compounds known to stimulate phagocyte-mediatedAGE-specific activity (see U.S. Pat. No. 4,665,192, issued May 12, 1987and U.S. Pat. No. 4,900,747, issued Feb. 13, 1990, and copending U.S.application Ser. No. 07/878,837, filed May 5, 1992).

Accordingly, the method of the present invention generally comprisesexposing brain tissue to AGE-amyloid polypeptide, or exposing pancreatictissue to AGE-amylin, which can result in activation of the mechanismsfor an increase in the recognition and removal of amyloid that hasundergone advanced glycosylation.

In a further embodiment, where a subject presently manifests thesymptoms of a disease associated with amyloidosis as described above,particularly involving dementia in neurodegeneration or adult onsetdiabetes, the present invention contemplates modifying the amyloidplaques to increase the level of AGEs, so as to increase theavailability of the plaques as targets for degradation by the pathwaysof recognition and removal of AGE-modified molecules, in particular byactivation of phagocytic cells. For example, amyloid targeting agents,like Congo Red and Thioflavin (see Caughey et al., U.S. Pat. No.5,276,059, issued Jan. 4, 1994, which is hereby incorporated byreference in its entirety), including derivatives and analogs thereofthat demonstrate affinity for binding amyloid, can be modified to bearAGEs or AGE precursors (hereinafter, both AGE and AGE precursor modifiedtargeting agents are termed “AGE” modified targeting agents) andadministered so as to target said AGEs or AGE precursors to amyloiddeposits. The structures of Thioflavin T and Congo Red are shown below:

The AGE-amyloid targeting agents of the invention can be administeredvia any route, e.g., i.v., i.p., i.m., intraventricularly,intracranially, orally, nasally, through a skin patch, etc. In aspecific aspect, these agents are modified to be capable of crossing theblood brain barrier, and thus modified, are attractive candidates forincreasing the level of AGEs on amyloid plaques associated withneurodegenerative disease.

Any AGE or AGE precursor, such as an Amadori compound, can be conjugatedto the amyloid targeting agent for use in increasing the level of AGEmodification of amyloid. Examples of such AGEs or AGE precursorsinclude, but are not limited to, FFI, fructopyranose and derivativesthereof, and the like. In specific embodiments, infra, Thioflavin isconjugated with an Amadori compound, such as6-amino(1-deoxy-β-D-fructopyranos-1-yl) and 6-N,N-dimethylamino(1-deoxy-β-D-fructopyranos-1-yl) groups as shown in anExample, infra, to form AGE-Thioflavin (AGE-TF). In another embodiment,Congo Red is conjugated with an Amadori compound, e.g., as outlined inScheme I, as follows:

The Thioflavin or Congo Red moiety targets the Amadori compound toamyloid plaques and fibrils, in order to induce AGE formation mediatedby the Amadori product on or in immediate association with the amyloidplaque or fibril, resulting in formation of AGE-modified amyloid, thusincreasing the likelihood of uptake of the amyloid plaque or fibril bylocal or recruited phagocytic cells.

In a specific embodiment, AGE-TF can be administered to an individualwith AD in order to induce AGE formation in the βAP-amyloid plaquesassociated with AD. Increased levels of AGE modification of the amyloidincreases the likelihood of uptake by central nervous system microgliaor recruited peripheral monocytes, or both, and facilitates removal ofthe amyloid.

In another specific embodiment, AGE-TF or AGE-Congo Red can beadministered to an individual with Type II diabetes to induce AGEformation of the amylin-amyloid plaques associated with Type IIdiabetes. Increased levels of AGE modification of the amyloid increasesthe likelihood of uptake by local or recruited phagocytic cells, andfacilitates removal of the amyloid.

The effectiveness of an AGE bearing targeting agent, such as AGE-TF orAGE-Congo Red, can be tested in vitro and in vivo for efficacy at AGEmodification of amyloid. This AGE modification may be non-covalentthrough the association of the targeting agent and amyloid, or covalentdue to the inherent reactivity of the AGE or AGE precursor for sites onthe amyloid. In the following examples, the amyloid polypeptide flAP andthe amyloid targeting agent Thioflavin (TF) are described forconvenience. However, the present disclosure contemplates performance ofthe same or similar assays with any amyloid model system and anyamyloid-specific targeting agent, and is not intended to be limited tothe following examples.

An in vitro assay can be used to determine the ability of AGE-TF to AGEmodify insoluble or aggregated βAP or amylin. For example, AGE-TF can beincubated with insoluble βAP or insoluble amylin to produce AGE-modifiedinsoluble or aggregated βAP or amylin. The level of AGE modification canbe determined, e.g., by ELISA using an anti-AGE antibody, and comparedto a control treated with the unmodified Thioflavin. Further testing forclearance of the AGE-modified insoluble βAP can be conducted byincubation with cultured phagocytic cells, such as mouse peritonealmacrophages, elicited macrophages, the RAW 264.7 cell line, humanperipheral monocytes, or inicroglia or astroglia primary cells or celllines.

Involvement of AGE-receptor-mediated uptake by phagocytic cells can beevaluated with a standard binding assay. Insoluble or aggregatedlabelled βAP (e.g., ¹²⁵I-βAP, although any labelling means known in theart, such as are discussed below, can be used) is contacted with anAGE-TF or TF alone (control), and incubated with about 106 cells perwell in the presence of increasing concentrations of cold AGE-BSA as astandard competitor. The amount of labelled βAP or amylin bound in theabsence of competing AGE-BSA is then compared to the amount in thepresence of different concentrations of AGE-BSA. Standard methods canthen be used to derive the number, affinity and association kinetics ofAGE-modified amyloid binding sites on the cells. Labelling of the βAP oramylin should precede AGE modification by AGE-TF treatment to ensurethat the βAP or amylin is the labelled moiety. Additional controls caninclude untreated βAP or amylin alone.

An uptake assay can also be performed. In the uptake assay, cells areincubated with medium containing AGE-TF treated, insoluble or aggregatedlabelled βAP or amylin versus control (e.g., labelled flAP or amylinalone, TF alone and/or TF treated labelled βAP or amylin) for varioustimes, ranging from 1-48 hours, e.g., 1, 2, 4, 24 and 48 hours, at 37°C. After the incubation, the cells are separated from the culture fluid.The content of label in the cell fraction and culture fluid fraction ismeasured, e.g., by TCA precipitation of the labelled compounds.Alternatively, wells can be pre-coated with the labelled test andcontrol compounds, in either their soluble or insoluble aggregatedforms, incubated with cells, and the presence of labelled compounds inthe cell culture fluid, which is indicative of degradation of thecompounds by uptake of the compounds coated on the wells, can beassayed. Preferably, the label is 125I or FITC, more preferably 125I.The uptake assay can be performed with the same cells as used for theclearance assay.

The invention also contemplates use of in vivo assays to demonstrate AGEmodification of amyloid. In one embodiment, AGE-TF or AGE-Congo Red canbe administered to female Syrian hamsters, which develop amyloidosis inthe liver, spleen and kidney within about one year after birth(resulting in a much shorter life expectancy than for male hamsters; seeCoe and Ross, 1985, J. Clin. Invest. 76:66-74), and in whichadministration of diethylstilbestrol (DES) accelerates this amyloidosis.To determine the effect of AGE-TF or AGE-Congo Red, a group (n=4 to 10)of 6-12 month old hamsters treated with DES at 3 months of age aretreated with 1-2 intraperitoneal injections of about 0.1 to about 1 mgof AGE-TF or AGE-Congo Red in an appropriate carrier, such as bufferedsaline, per week for varying periods of time. Control animals receiveinjections of the carrier, AGE alone, and TF or Congo Red alone. Thepresence and level of AGE-modification of amyloid deposits can be testedby the tissue squash method (Coe and Ross, 1990, J. Exp. Med.171:1257-67) and ELISA (as described herein) after varying periods oftime, e.g., 1 month, 2 months, and 4 months.

A similar in vivo assay can be performed on mice or hamsters inoculatedintraperitoneally or intracranially with scrapie. About 1 month to 12months, preferably 1 month to about 6 months, after infection withscrapie, the animals can be treated with AGE-TF or AGE-Congo Red, e.g.,weekly or biweekly with intraperitoneal or intracranial injections ofabout 0.1 to about 1 mg of AGE-TF or AGE-Congo Red. Tissue samples orsections of affected organs (spleen for intraperitoneal infection, brainfor intracranial infection) can be obtained. The presence of amyloid inspleen or brain can be detected histologically or immunochemically(immunohistologically or by ELISA with an anti-PrP antibody). The levelof AGE modification of the tissue can be detected immunohistologicallyor by ELISA, e.g., using the anti-AGE-RNase antibody as describedherein.

Such an experiment can also be performed with cats, rats, or mice thatare genetically predisposed or treated to develop a Type II diabeticcondition. About 1 month to 12 months, preferably 1 month to about 6months, after diabetes onset, the animals can be treated with AGE-TF orAGE-Congo Red, e.g., weekly or biweekly with intraperitoneal orintravenous injections of about 0.1 to about 1 mg of AGE-TF or AGE-CongoRed. Tissue samples or sections of pancreas, particularly areascontaining islet cells, can be obtained. The presence of amyloid can bedetected histologically or immunochemically (immunohistologically or byELISA). The level of AGE modification of the tissue can be detectedimmunohistologically or by ELISA, e.g., using the anti-AGE-RNaseantibody as described herein.

The effectiveness of AGE modification of amyloid in inducing removal ofthe amyloid can be determined by detecting the amount of amyloid inaffected tissues and comparing that amount to the amount in controlanimals after various periods of time. According to this aspect of theinvention, the time course of pathology and treatment can involveamyloidosis, AGE-modification of amyloid, and clearance of theAGE-modified amyloid. Thus, the presence of amyloid and the level of AGEmodification of the amyloid will depend on the point within the timecourse at which the sample is obtained for testing. The time course canbe readily determined by obtaining and assaying samples at varioustimes.

In a further embodiment, the method of inducing AGE modification of theamyloid can be combined with the methods discussed above for activatingmechanisms for the recognition and clearance of AGE-modified amyloidplaques.

Diagnostic Methods

The present invention also relates to a method for detecting thepresence of or monitoring the course of a disease or disorder associatedwith amyloidosis comprising detecting the presence of or measuring thelevel or amount of an AGE-amyloid polypeptide that is found in theamyloid plaques characteristic of such a disease. Detecting the presenceof AGE-amyloid polypeptides can indicate the existence of the diseasecondition, and thus may be useful alone or in conjunction with othercriteria in diagnosis of such a disease. An increase in the level ofAGE-amyloid polypeptide compared to the level detected in the subject atan earlier time, or to the level found in normal individuals, canindicate disease progression; a decrease in the level compared to thelevel in the subject at an earlier time, or the level found in normalindividuals, can indicate regression of the disease.

In a specific aspect, the invention provides for diagnosing ormonitoring the course of a neurodegenerative disease associated withamyloidosis in mammals, by measuring the corresponding presence andamount or level of AGE-βAP. AGE-βAP can be detected in biological fluidssuch as, but not limited to, blood, plasma, serum, urine, cerebrospinalfluid, and the like. Alternatively, the sample can be ahistopathological sample, such as a biopsy or tissue sample.

In another embodiment, the invention relates to a method for detectingthe presence of or monitoring the course of Type II diabetes bymeasuring the corresponding presence or amount or level of AGE-amylin.AGE-amylin can be detected in biological fluids, such as, but notlimited to, blood, plasma, serum, urine, peritoneal fluid, and the like.Alternatively, the sample can be a histopathological sample, such as abiopsy or tissue sample.

The presence or level of AGEs may be followed directly by assaytechniques such as those discussed herein, through the use of anappropriately labeled quantity of at least one of the binding partnersto AGE-amyloid polypeptide as set forth herein. Alternately, AGEs can beused to raise binding partners or antagonists that could in turn, belabeled and introduced into a medium to test for the presence and amountof AGEs therein, and to thereby assess the state of the host from whichthe medium was drawn.

The term “ligands” includes such materials that would bind toAGE-amyloid peptide-binding partners, and would include such materialsas are prepared by the reaction of AGE-flAP or AGE-amylin with avidin orbiotin, or the preparation of synthetic AGE-flAP or AGE-amylinderivatives that may be prepared from the reaction of βAP or amylin withreducing sugars such as glucose, glucose-6-phosphate (G-6-P), fructoseor ribose, and AGE-fAP or AGE-amylin conjugation with peptides, proteinsand other biochemicals such as bovine serum albumin (BSA), avidin,biotin derivatives, and enzymes such as alkaline phosphatase. Likewise,enzymes and other carriers that have undergone advanced glycosylationmay also serve as ligands in any of the assays of the present invention.Accordingly, carriers such as carbohydrates, proteins, syntheticpolypeptides, lipids and biocompatible natural and synthetic resins, andany mixtures of the same may be reacted with sugars to form advancedglycosylation endproducts and may thereby be useful in the presentmethods. The present diagnostic methods are intended to contemplate allof the foregoing materials within their scope.

The term “AGE binding partners” is intended to extend to anti-AGEantibodies and to other cellular AGE binding proteins or receptors forAGEs, which AGEs may be found on peptides, molecules and cells. Aparticular AGE binding partner is an anti-AGE antibody raised in rabbitsand isolated therefrom for use as contemplated herein.

Thus, both AGE-βAP or AGE-amylin and any binding partners thereto thatmay be prepared, are capable of use in connection with variousdiagnostic techniques, including immunoassays, such as aradioimmunoassay, using for example, a receptor or other ligand to anAGE that may either be unlabeled or if labeled, e.g., by radioactiveaddition or radioiodination.

In an immunoassay, a control quantity of a binding partner to AGE-βAP orAGE-amylin may be prepared and optionally labeled, such as with anenzyme, a compound that fluoresces and/or a radioactive element, and maythen be introduced into a tissue or fluid sample of a mammal. After thelabeled material or its binding partner(s) has had an opportunity toreact with sites within the sample, the resulting mass may be examinedby known techniques, which may vary with the nature of the labelattached.

The labels most commonly employed for these studies are radioactiveelements, enzymes, chemicals which fluoresce when exposed to ultravioletlight, and others.

Suitable examples of radioactive elements include ³H, ¹⁴C, ³²p, ³⁵S,³⁶Cl, ⁵¹Cr, ⁵⁷Co, ⁵⁸Co, ⁵⁹Fe, ⁹⁰Y, ¹²⁵I, ¹¹³I, and ¹⁸⁶Re. In theinstance where a radioactive label, such is prepared with one of theabove isotopes is used, known currently available counting proceduresmay be utilized.

In the instance where the label is an enzyme, detection may beaccomplished by any of the presently utilized colorimetric,spectrophotometric, fluorospectro-photometric, thermometric,amperometric or gasometric techniques known in the art. The enzyme maybe conjugated to the advanced glycosylation endproducts, their bindingpartners or carrier molecules by reaction with bridging molecules suchas carbodiimides, diisocyanates, glutaraldehyde and the like.

Many enzymes which can be used in these procedures are known and can beutilized. The preferred are peroxidase, 6-glucuronidase,β-D-glucosidase, β-D-galactosidase, urease, glucose oxidase plusperoxidase, hexokinase plus GPDase, RNAse, glucose oxidase plus alkalinephosphatase, NAD oxidoreductase plus luciferase, phosphofructokinaseplus phosphoenol pyruvate carboxylase, aspartate aminotransferase plusphosphoenol pyruvate decarboxylase, and alkaline phosphatase. U.S. Pat.Nos. 3,654,090; 3,850,752; and 4,016,043 are referred to by way ofexample for their disclosure of alternative labeling material andmethods. A particular enzymatic detecting material is anti-rabbitantibody prepared in goats and conjugated with alkaline phosphatasethrough an isothiocyanate.

A number of fluorescent materials are known and can be utilized aslabels. These include, for example, fluorescein, rhodamine and auramine.A particular fluorescent detecting material is anti-rabbit antibodyprepared in goats and conjugated with fluorescein through anisothiocyanate.

In addition to therapeutic uses, the antibodies of the invention can beused to detect AGE-amyloid polypeptides in amyloid or in solution. Inparticular, antibodies of the invention can be used to determine theamount and location of the AGE in amyloid plaques in the mammalian body.For convenience, the antibody(ies) to the AGE will be referred to hereinas Ab, and antibody(ies) reactive with Ab₁ as Ab₂.

The amount of AGE-amyloid polypeptide in a biological fluid or thedegree of advanced glycosylation in amyloid plaques can be ascertainedby the usual immunological procedures applicable to such determinations.A number of useful procedures are known. Three such procedures which areespecially useful utilize either the AGE-amyloid polypeptide labeledwith a detectable label, antibody Ab, labeled with a detectable label,or antibody Ab₂ labeled with a detectable label. The procedures andtheir application are all familiar to those skilled in the art andaccordingly may be utilized within the scope of the present invention.An example of a “competitive” procedure is described in U.S. Pat. Nos.3,654,090 and 3,850,752. An example of a “sandwich” procedure, isdescribed in U.S. Pat. Nos. RE 31,006 and 4,016,043. Still otherprocedures are known such as the “double antibody”, or “DASP” procedure.

In each instance, the AGE-modified substance forms complexes with one ormore antibody(ies) or binding partners and one member of the complex islabeled with a detectable label. The fact that a complex has formed,and, if desired, the amount thereof, can be determined by known methodsapplicable to the detection of labels.

It will be seen from the above, that a characteristic property of Ab2 isthat it will react with Ab₁. This is because an antibody raised in themammalian species in which Ab₁ was raised has been used in anotherspecies as an antigen to raise the antibody Ab₂. For example, Ab₁ may beraised in rabbits and Ab₂ may be raised in goats using a rabbit Ab as anantigen. Ab₂ therefore would be anti-rabbit antibody raised in goats.

Accordingly, a test kit may be prepared for the demonstration ofAGE-amyloid polypeptide in a sample, comprising:

(a) a predetermined amount of at least one labeled immunochemicallyreactive component obtained by the direct or indirect attachment ofAGE-amyloid polypeptide or an AGE binding partner to a detectable label;

(b) other reagents; and

(c) directions for use of said kit.

More specifically, the diagnostic test kit may comprise:

(a) a known amount of an AGE, such as AGE-BSA, AGE-βAP, or AGE-amylin(or a binding partner thereof) generally bound to a solid phase to forma immunosorbent, or in the alternative, bound to a suitable tag, orplural such components, etc. (or their binding partners) one of each;

(b) if necessary, other reagents; and

(c) directions for use of said test kit.

By example, a solid phase assay system or kit may comprise the solidsubstrate with either bound binding partner and labeled AGE-amyloidpolypeptide or bound AGE-amyloid polypeptide and labeled bindingpartner. A sample to be assayed is then placed in contact with the boundand unbound reagent. A competitive reaction between the labeled materialand any unlabeled binding partner(s) in the sample will cause theretention of a dependent quantity of the former on the solid substrate,whereupon it can be precisely quantitatively identified. The foregoingexplanation of a particular competitive assay system is presented hereinfor purposes of illustration only, in fulfillment of the duty to presentan enabling disclosure of the invention. It is to be understood that thepresent invention contemplates a variety of diagnostic protocols withinits spirit and scope.

In a preferred aspect of the invention, the AGE assay described inMakita et al. (1992, J. Biol. Chem. 267:5133-38) is used to detect thepresence and determine the amount of AGE-amyloid in a tissue sample,particularly a sample that contains amyloid, or the amount of AGE-βAP,AGE-scrapie protein, or AGE-amylin present in a sample.

In a specific embodiment, infra, antibodies reactive with AGEs are usedto detect increased levels of AGE-proteins in brain tissue fromindividuals diagnosed with AD compared to normal individuals.

In another specific embodiment, antibodies to PrP (the scrapie or prionprotein; e.g., Kascsak et al., 1987, J. Virol. 61:3688-3693) andantibodies to AGE can be used to show co-localization of these epitopesin a tissue sample. For example, brain sections from hamsters or miceinfected with scrapie can be immunohistochemically stained with rabbitpolyclonal anti-PrP, anti-AGE-RNase, and a control anti-RNase. Inscrapie (a subacute spongiform encephalopathy), the characteristicspongiform encephalopathy is characterized by PrP-associated lesions,which contain amyloid deposits. These immunochemical studies can showthat within a single scrapie diseased brain, PrP and AGEs co-localize inthe amyloid of these lesions.

The invention may be more completely understood by reference to thefollowing non-limiting examples, which are provided solely as exemplaryof specific embodiments of the invention.

EXAMPLE 1 Age-Amyloid in Alzheimer's Disease

Alzheimer's disease (AD) is characterized by deposits of aggregatedβ-amyloid peptide (βAP) in the brain and cerebrovasculature. After aconcentration-dependent lag period during in vitro incubations, solublepreparations of synthetic βAP slowly form fibrillar aggregates thatresemble natural amyloid and are measurable by sedimentation orThioflavin-T-based fluorescence. Aggregation of soluble βAP in these invitro assays is enhanced by addition of small amounts of pre-aggregatedβ-amyloid “seed” material. These seeds have also been prepared hereinusing a naturally occurring reaction between glucose and protein aminogroups resulting in the formation of advanced glycosylation endproducts(AGEs) which chemically crosslink proteins. AGE-modified βAP-nucleationseeds further accelerated aggregation of soluble βAP compared tonon-modified “seed” material. Over time, nonenzymatic advancedglycosylation also results in the gradual accumulation of a set ofpost-translational covalent adducts on long-lived proteins in vivo.Using a standardized competitive ELISA assay, plaque fractions of ADbrains were found to contain about 3-fold more AGE adducts per mgprotein than found in like preparations from healthy, age-matchedcontrols. These results indicate that the in vivo half-life of B-amyloidis prolonged in AD, resulting in greater accumulation of AGEmodifications, which in turn can act to promote accumulation ofadditional amyloid.

Materials and Methods

Aggregation and Seeding Reactions.

Synthetic, HPLC-purified peptides representing the first 28 (βAP 1-28)and the first 40 amino acids (βAP 1-40) of the 42 amino acid βAP wereobtained from Bachem (Torrance, Calif.). Aggregation of soluble βAP 1-28or 3AP 1-40 at different concentrations was initiated by addition of 0.1M sodium acetate (NaOAc) at the indicated pH (between 4.7 and 7.5) andcontinued for the indicated times. Quantitative aggregate formation withsub-millimolar βAP concentrations was detected using the procedure ofLeVine (1992, Protein Science 2:404-410). Briefly, fluorescence ofaggregates added to 10 μM Thioflavin-T (Aldrich)/50 mM potassiumphosphate buffer, pH 6.0, was measured upon excitation at 450±5 nm anddetection of emission at 482±10 nm on a Perkin Elmer LS-50Bspectrofluorimeter. Where indicated, small amounts of pre-formedaggregates or “nucleation seeds” were added to the soluble βAP andaggregation initiated with 0.1 M sodium acetate.

Generations of “Seeds”.

Soluble βAP 1-40 (250 μM) and 0.2 M sodium phosphate buffer, pH 7.5,were incubated with or without 1 M glucose at 37° C. to generatepre-formed aggregates of AGE-βAP referred to as “AGE-βAP seed” or “βAPseed”, respectively. After incubation, protein concentrations of seedpreparations were measured and adjusted with buffer to 150 μM finalconcentration. Using competitive ELISA (Makita et al., supra), AGE-βAPseed contained 50 AGE Units/mg protein and βAP seed contained less than0.5 AGE Units/mg protein. Lysines at positions 16 and 28 of βAP containprimary amino groups which may react with reducing sugars to generateadvanced glycosylation endproducts of AGEs. Indicated amounts of glucoseand/or aminoguanidine, a potent inhibitor of advanced glycation andcrosslink formation (Brownlee et al., 1986, Science 232:1629-1632), werealso added to solutions of βAP before sodium acetate in someexperiments.

Aggregation of βAP at lower (physiological) concentrations wasquantitated by the method of Burdick et al. (1992, J. Biol. Chem.267:546-554). Synthetic preparations of βAP 1-40 were labeled with 1251(NEN) and chloramine-T (Sigma) for 1 minute before the reaction wasquenched with 10 mM tyrosine and sodium meta-bisulfite. Unincorporatedlabel was removed by filtration through a SEPHADEX G-10 columnequilibrated in 0.5×phosphate buffered saline (PBS), pH 7.4. The¹²⁵I-labeled βAP (approximate specific activity of 3×10⁶ cpm/μg) wasimmediately diluted to 5 riM final concentration in the presence ofvarious “seeds,” glucose and/or aminoguanidine at the indicatedconcentrations. After various incubation periods at 37° C., aggregationreactions were underlayed with 20% sucrose/0.1 M sodium acetate at thesame pH as the incubation mixture, centrifuged for 30 minutes at50,000×g, and frozen in liquid nitrogen. Each microfuge tube was cut andthe bottom 5 mm representing the aggregated sedimentable fraction whichhad pelleted through the sucrose cushion, was counted in a gammacounter. The remainder of the tube and liquid were also counted. Theamount of aggregate formed was calculated as a percentage equal to thenumber of counts in the pellet divided by the total number of counts pertube (pellet +remainder) multiplied by 100.

Measurement of AGEs with Competitive ELISA.

Aliquots of frozen pre-frontal cortex (Brodman areas 9 and 10) frompatients with and without behaviorally and neuropathologically confirmedAD were resuspended in 10 volumes per wet weight of 2% sodiumdodecylsulfate (SDS)/0.1 M B-mercaptoethanol (ME), and Douncehomogenized. The homogenate was boiled for 10 minutes and thencentrifuged at 10,000×g for 10 minutes. Supernatants were aspirated andthe resulting pellets washed three times with PBS at 10,000×g for 10minutes. In some experiments, this crude plaque fraction was furtherwashed twice with 4 M urea and twice more with PBS before proteasedigestion. PBS-washed pellets were resuspended in one-tenth the originalhomogenate volume of PBS and 0.1% Proteinase-K (Boehringer Mannheim),digested overnight at 37° C. and heat inactivated at 75° C. for 3 hours.Quadruplicate 5, 10 and 20 μl aliquots of plaque-containing pelletfractions were assayed for AGE content using a competitive ELISA (Makitaet al., 1992, J. Biol. Chem. 267:5133-38), against standardizedpreparation of AGE-modified bovine serum albumin (AGE-BSA). Only valuesin the linear range of the standard curve were included in the analyses.

Protein amounts were quantitated with micro-BCA kit (Pierce) and withfluorescamine (Bohlen et al., 1973, Arch. Biochem. Biophys.155:213-220). AGE Units were interpolated from a standard dilution curveof AGE-BSA and divided by the sample protein concentration to give AGEUnits per mg protein. Statistical analysis using Student's test wasperformed with the StatWorks program for Macintosh using a Macintoshpersonal computer (Apple Computer, Inc.).

Results

βAP Aggregation. Displays Nucleation Dependent Kinetics.

Aggregation of synthetic βAP was analyzed in vitro where the aggregationrate of soluble βAP was found to depend mainly upon pH and the initialconcentration. The kinetics of aggregation were determined empirically.600 μM βAP 1-28 spontaneously and rapidly formed Thioflavin-Tfluorescent aggregates within minutes at pH 7.2 (FIG. 1). Atconcentrations below 300 AM, however, βAP aggregated very slowly with aconsiderable lag before fluorescent aggregates were measurable.

At low concentrations of βAP, the lag period preceding measurableaggregation is reminiscent of crystallization reactions, in whichprotein solutions very slowly assume a single conformation and aggregatein a well-defined, molecular packing arrangement (Jarrett et al., 1993,Biochemistry 32:4693-4697; Jarrett and Lansbury, 1993, Cell73:1055-1058; Come et al., 1993, Proc. Natl. Acad. Sci. USA90:5959-5963). The kinetics of such aggregation in many cases can besignificantly accelerated by the addition of a pre-formed aggregate or“seed” material as nucleation centers for additional aggregateformation. Stable “βAP seed” material was prepared by incubating 250 AMβAP 1-40 for 4 months at 37° C. Compared to the small amounts offluorescent aggregate measured when 300 μM soluble βAP or 75 μM “βAPseed” were separately incubated in control preparations, co-incubationof soluble βAP plus “βAP seed” resulted in the progressive accumulationof much larger amounts of fluorescent aggregates (FIG. 2).

AGE-modified βAP seed was also prepared by incubating soluble βAP in 1 Mglucose/pH 7.5 phosphate buffer at 37° C. for 4 months (“AGE-βAP seed”).AGE formation was confirmed by competitive ELISA where “βAP seed”contained less than 0.5 AGE Units/mg protein and “AGE-βAP seed”contained 50 AGE Units/mg protein, which is comparable, within an orderof magnitude, to the AGE content of plaques from AD brains (see below).When 75 μM “AGE-βAP seed” was co-incubated with 300 μM soluble βAP, muchmore fluorescent aggregate was detected than in parallel incubationswith unmodified “βAP seed” and soluble βAP (FIG. 2). As with “βAP seed,”separate incubation of “AGE-βAP seed” alone did not lead to a change inthe small amount of fluorescent aggregate with time.

To test whether this seeding phenomenon occurs at concentrations of βAPthat are typically found in vivo, a sedimentation assay was employed tomeasure aggregation. Aggregation of 10 nM solutions of soluble¹²⁵I-labelled synthetic βAP 1-40 in 0.1 M sodium acetate, pH 7.0,increases slowly over a two-day incubation (FIG. 3). If 10 nM labeledRAP was co-incubated with 200 nM unlabeled “βAP seed” material, theamount of sedimentable label was similar to the no seed, soluble βAPonly curve. In contrast, co-incubation of 10 nM soluble βAP with 200 nM“AGE-βAP seed” increased the amounts of sedimentable label compared tothe “βAP seed” and no seed experimental points. Thus, the amount oflabeled βAP associated with sedimentable aggregates observed inco-incubations of soluble βAP and “AGE-βAP seed” is greater than thatformed in co-incubations with “OAP seeds” or no seed under conditions inwhich pH and soluble βAP concentrations are physiological.

Glucose Modifies Kinetics of Aggregation.

In the process of advanced glycosylation, formation of a Schiff basebetween glucose and a protein amino group precedes subsequent maturationof Amadori products into AGE-modified βAP. Aminoguanidine is a compoundwhich prevents AGE formation following initial glucose reactions withprotein amines (Brownlee et al., 1986, Science 232:1629-1632).Co-incubation of 400 tM βAP 1-28 in 0.1 M sodium acetate, pH 7.0, with100 mM glucose stimulates fluorescent aggregate formation when comparedto parallel incubations of soluble βAP, soluble βAP+AG, or solubleβAP+AG+glucose (FIG. 4). Separate incubations of glucose or AG failed togenerate detectable fluorescent signals.

AGE Content of Brain Fractions.

On average, plaque-enriched fractions isolated from samples ofpre-frontal cortex of 10 AD brains contained significantly more AGEmodifications than did corresponding preparations from 7 healthycontrols (8.9±1.4 versus 2.7±0.5 AGE Units/mg protein, p=0.002, see FIG.5). The average chronological age of both the AD and control groups was77 years. The presence of AGEs in similar fractions of parietal cortexfrom AD and normal brains has also been observed (data not shown).Supernatant fractions of SDS-soluble proteins from AD and control brainsroutinely contained less than 0.1 AGE Units/mg protein.

Discussion

Although advanced glycosylation adducts form spontaneously in vivo,their accumulation is slow and becomes most notable with increasing timeon long-lived tissue components. Along with time and the availability ofsusceptible protein amino groups, ambient glucose is the other majordeterminant of AGE formation. Thus, quantitative analysis of the degreeof AGE modification of a single protein species under standardizedglycemic conditions yields an index of the protein's half-life in vivo.It was found that plaque-enriched fractions isolated from AD brainsamples contained about 3-fold more AGE modifications than didcomparable fractions prepared from age-matched control brains,suggesting that RAP half-life is prolonged in AD. That amyloidcomponents exhibit a prolonged half-life in AD is also supported bystudies that demonstrated the time-dependent, nonenzymatic isomerizationof aspartyl-residues occurring at positions 1 and 7 of βAP isolated fromAD brain (Roher et al., 1993, J. Biol. Chem. 268:3072-3083), althoughthis study latter did not include normal controls.

AD is characterized by progressive dementia and increased numbers andamount of amyloid plaques compared to healthy age-matched controls.While a causal relationship between increased dementia and plaquenumbers has not been proven, the gradual onset of symptoms appears toparallel the progressive deposition of β-amyloid. From in vitro studies,it is clear that millimolar concentrations of soluble βAP willspontaneously aggregate into fibrillar amyloid structures following anucleation-dependent mechanism. At lower concentrations, the requirementfor nucleus formation introduces a substantial lag period during which asolution of βAP that still requires most of this time to formaggregation nuclei is indistinguishable from one on the verge of rapidaggregation and growth into a “one dimensional crystal” (Jarrett andLansbury, 1993, Cell 73:1055-1058). The effect of thisconcentration-dependent nucleus formation is extreme as illustrated bypublished calculations that show an APP mutation in a Swedish form offamilial AD which raises soluble βAP concentrations 6-fold (Cai et al.,1992, Science 259:514-516; Citron et al., 1992, Nature 360:672-674)should reduce the lag time before aggregate growth occurs from 100 yearsto about 3 hours (Jarrett et al., 1993, Biochem. 32:4693-4697). Sincethe cerebrospinal fluid concentration of soluble βAP in AD patients isthe same as in age-matched controls (Oosawa et al., 1993, Soc. Neurosci.Abs. 19:1038; Shoji et al., 1992, Science 258:126, 129), the rate ofconcentration-dependent self-formation of nuclei, as reflected by theamounts of amyloid formed and deposited, might also be expected to bethe same. As this latter similarity is not observed and AD brains formsubstantially more deposits of aggregated βAP than their non-diseasedcounterparts, then it appears that the increased amount of amyloidpresent in afflicted brain tissue results, at least in part, from moreefficient nucleated aggregation than occurs in healthy brain parenchyma.

For purposes of clarifying the operation and discovery of the invention,but without intending to be limited to any particular theory orhypothesis by way of this explanation, nucleation seeds can be thoughtof as structures of βAP possessing a specific conformation that promotesthe rapid accretion of additional soluble βAP resulting in the growth ofinsoluble βAP aggregates. Since the spontaneous formation of nuclei isthermodynamically unfavorable (Jarrett et al., 1993, supra), processesknown to chemically crosslink proteins in vivo might serve to stabilizespecific conformations of βAP with nucleating characteristics. Advancedglycosylation is a naturally occurring process of covalentpost-translational modification of proteins that readily occursextracellularly. AGEs in other contexts are well-known asprotein-protein crosslinking agents in vivo and in vitro, and AGEaccumulation on matrix proteins is associated with increased resistanceto proteolysis (Bucala et al., 1992, in Post-Translational Modificationsof Proteins, Harding et al., Eds., CRC Press, Boca Raton 2:53-79).

At physiological concentration and pH in vitro, soluble βAP aggregatesslowly. Of note, the addition of preformed aggregates ofAGE-modified-βAP stimulated markedly more rapid aggregation of nMsolutions of soluble βAP than did preformed aggregates of unmodified βAPin two-day incubations. That this acceleration occurred at normal pH,physiological concentrations of soluble βAP, and with seeds containingamounts of AGE-modifications comparable to those found in AD plaquefractions, suggests that a similar process could occur in vivo.

Glycation adducts comprise a structurally heterogeneous family ofproducts that slowly evolve chemically by a variety of rearrangement,condensation and elimination reactions. The particular AGE species thatenhance nucleation remain unknown, but these may be relatively earlyglycosylation products, as evidenced by the time course over whichglucose accelerated the rate of fluorescent βAP aggregate formationcompared to aggregation in the presence of glucose and aminoguanidine, aspecific inhibitor of AGE formation. Recognizing that plaque numbersincrease in association with neuronal degeneration and cognitive declinein AD, and that aggregated but not soluble βAP is actively neurotoxic(Pike et al., 1991, Eur. J. Pharm. 207:367-368; Pike et al., 1993, J.Neuroscience 13:1676-1687), interference with the processes by which AGEformation enhances βAP aggregation can provide new therapeuticopportunities to reduce the pathophysiological changes associated withAD.

EXAMPLE 2 Co-Localization of Ages and Prion Protein H

amsters were infected by intracerebral injection with a strain ofhamster-adapted murine scrapie. After 300 days, the hamsters weresacrificed, the brains sectioned, and the sections fixed on microscopeslides. The fixed sections were treated with 70%. formic acid for 10minutes and washed. The slides were then reacted with rabbit antiseraspecific for RNase (control antisera), prion protein (PrP; Kascsak etal., 1987, J. Virol. 61:3688-93), and AGE (anti-AGE-RNase antisera, asdescribed in Makita et al., 1992, J. Biol. Chem. 267:5133-38). Eachserum was diluted 1:500 prior to incubation with the tissue samples. Thereactions were incubated overnight at 4° C. Following reaction with therabbit antisera, the samples were washed with PBS and reacted with analkaline phosphatase (AP)-conjugated anti-rabbit antibody. The sampleswere developed with a fuschin AP substrate (Dako), which produces a redcolor.

The results of this experiment are shown in FIG. 6. The histologicalslides show regions of PrP-associated plaques identified with theanti-PrP antiserum (FIG. 6B). PrP is the purified scrapie protein thatacts as the infectious agent, and that is associated with amyloiddeposition in affected subjects. The anti-AGE antiserum generatedagainst AGE-RNase also decorated the amyloid plaques. A controlantiserum (anti-RNase) did not react with the histological samples.

These results indicate that the AGEs are present in the amyloid plaquesthat are characteristic of spongiform encephalopathy. As the scrapieamyloid plaque forms, it acquires AGE modifications that are detectableby antibodies. AGE modification of the scrapie amyloid plaque can occurthrough AGE modification of soluble PrP or the amyloid plaque itself, orby both mechanisms.

EXAMPLE 3 Age-Modified Thioflavins AGE-Thioflavin A, 3

2-(4-[([(6-aminohexyl)amino]carbonyl)amino]phenyl)-6-methylbenzothazole,1, was prepared by combining 2-(4-amniophenyl)-6-methylbenzothiazole(0.48 g, Aldrich Chemical Company) with bis(trichloromethyl) carbonate(0.22 g) in xylene (10 ml), and heating the mixture at reflux for 3 hr.The not clear orange supernatant was decanted from some insolublematerial and cooled, giving an orange suspension of2-(4-isocyanatophenyl)-6-methylbenzothiazole. To this suspension wasadded 6-(t-butoxycarbonylamino)-1-hexylamine (0.40 g) in dichloromethane(10 ml). The mixture was stirred for 2 hr at room temperature (RT),forming a buff-colored suspension. The suspension was filtered and thesolid washed with dichloromethane and t-butyl methyl ether to give 1 asan off-white powder, m.p. 164-165° C. This material (300 mg) wasdissolved in 3 ml of 1:1 trifluoroacetic acid:dichloromethane andstirred for 4 hr at room temperature. The solvent was removed and thesolid was partitioned between aqueous 0.1 N NaOH and dichloromethane.The organic layer was dried over sodium sulfate, filtered andconcentrated. The residue was triturated with t-butyl methyl ether andfiltered to yield 0.197 g of 1 as a white powder, m.p. 320° C.

(4-[([(6-[(1-deoxy-2,3:4,5-di-O-isopropylidene-β-D-fructopyranos-1-yl)amino]hexyl)amino]carbonyl)amino]phenyl)-6-methylbenzothazole,2, was prepared by dissolving 1 (0.196 g) and2,3:4,5-bis-O-(1-methylethylidine)-aldehydo-β-D-arabino-hexos-2-ulo-2,6-pyranose(0.30 g, see 1987, Carbohydrate Research 167:123-130) in 8:1methanol:water (4.5 ml) and treating the mixture with sodiumcyanoborohydride (0.063 g) and acetic acid (0.040 ml). The resultingsolution was stirred at 70° C. in an open flask for 6 hr. On cooling, awhite solid separated. Filtration gave 0.276 g of 2 as a white solid,m.p. 144-146° C.

(4-[([(6-[(1-deoxy-β-D-fructopyranos-1-yl)amino]hexyl)amino]carbonyl)amino]-phenyl)-6-methylbenzothiazole,3, was prepared by dissolving 2 (0.10 g) in 1,4-dioxane (4 ml), andtreating the solution with water (4 ml) and conc. HCl (0.5 ml) withstirring at room temperature for 24 hr. Triethylamine (1 ml) was thenadded to the yellow solution, giving a turbid colorless mixture whichwas triturated and stored at 4° C. The resulting white powderyprecipitate was filtered out and characterized as the 2,3-monoacetonide.The monoacetonide (0.025 g) was dissolved in 1.1 ml of 1.2 N HCl andstirred at 55° C. for 24 hr. Solid NaHCO₃ was added until the yellowcolor disappeared. Dichloromethane (1 ml) was added, and the mixture wasstirred until a white powder separated. Filtration gave 15 mg of 3.

AGE-Thioflavin B. 8

2-[4-(4-phthalimidobutyl)aminophenyl]-6-methylbenzothiazole, 4, wasprepared by dissolving 2-(4-aminophenyl)-6-methylbenzothiazole (0.139 g)and N-(4-bromobutyl)phthalimide (0.082 g) in dimethylforamide (DMF, 5ml) and heating at reflux under nitrogen for 4 hr. The solution wascooled and treated with 5 ml of water. The yellow precipitate wasfiltered out and recrystallized from dichloromethane to give 0.090 g of4, m.p. 169-170° C.

2-[4-(4-aminobutyl)aminophenyl]-6-methylbenzothiazole, 5, was preparedby heating 4 (0.20 g) with hydrazine hydrate (0.15 ml) in ethanol (20ml) at 60° for 4 hr. Hydrochloric acid (1 ml) was added and the solutionwas heated at reflux for 1 hr. After cooling, phthalhydrazide wasfiltered out and the filtrate was concentrated, diluted with water, andneutralized with NaHCO₃. The precipitate was filtered and dried to give5 in quantitative yield, m.p. 137-138° C.

(4-[(4-[(1-deoxy-2,3:4,5-di-O-isopropylidene-β-D-fructopyranos-1-yl)amino]butyl)amino]phenyl)-6-methylbenzothiazole,6, was prepared by refluxing 5 and 2,3:4,5-bis-O-(1-methylethylidine)-aldehydo-β-D-arabino-hexos-2-ulo-2,6-pyranose (1.0 g)in methanol for 3 hr. The solution was cooled and sodiumcyanoborohydride (0.11 g) was added. The mixture was then refluxed for 2hr. After cooling, the solvent was evaporated and the residue waspartitioned between water and dichloromethane. The organic layer waswashed with water, dried over sodium sulfate, and evaporated. Theresidue was chromatographed on silica gel using 20% ethyl acetate indichloromethane. The product fractions were concentrated to a gum whichcrystallized on trituration with methanol. Filtration gave 0.445 g of 6as a white solid, m.p. 129° C.

(4-[N-(4-[N-(1-deoxy-β-D-fructopyranos-1-yl)amino]butyl)-amino]phenyl)-6-methylbenzothiazolechloride, 6a, was prepared by dissolving 7 (0.025 g) in 8 ml 1:1methanol/water containing 1.3 mL conc. HCl and stirring at RT undernitrogen for 24 hours. The solution was neutralized with aq. NaHCO₃,concentrated to 2 mL, filtered to remove precipitated salt, and purifiedby HPLC on a preparative C-18 reverse phase silica gel column using agradient of 70-100% methanol in water. Compound 6a was collected in thepeak of retention time of 23.1 minutes. Proton NMR (CD₃OD) δ 1.66 (br,CH ₂CH ₂CH₂NHCH₂), 2.46 (s, CH ₃Ar), 2.68 (m, CH₂CH ₂NHCHD, ca. 2.84(2d, CH₂CH₂NHCH ₂), 3.8 (t, ArNHCH ₂), 3.55-4.05 (several m, 5H,carbohydrate C3-C6 protons), 6.70 (d, 2 aryl H ortho to NH), 7.28 (d,benzothiazole C5-H), 7.71 (s, benzothiazole C7—H), 7.76 (d,benzothiazole C4-H), 7.80 (d, 2 aryl H meta to NH).

(4-[N-(4-[N-(1-deoxy-2,3:4,5-di-O-isopropylidene-β-D-fructopyranos-1-yl)-N,N-dimethylammonio]butyl)-amino]phenyl)-6-methylbenzothiazoleiodide, 7, was prepared by dissolving 6 (0.020 g) in acetone (2 ml),containing KHCO₃ (8 mg) and treating the solution with methyl iodide(0.200 ml) at RT overnight. The mixture was filtered and the filtratewas evaporated to give 7 as a yellow solid in quantitative yield.Recrystallization from dichloromethane/ether gave yellow crystals, m.p.142° C.

(4-[N-(4-[N-(1-deoxy-β-D-fructopyranos-1-yl)-N,N-dimethylammonio]butyl)amino]phenyl)-6-methylbenzothiazolechloride, 8, was prepared by dissolving 7 (0.050 g) in 20 ml 1:1methanol/water containing 3.5 ml conc. HCl and heating at 50° C. undernitrogen for 18 hr. The solution was neutralized with aq. NaHCO₃,concentrated to 5 ml, and filtered to remove precipitated salt. Of this2 ml was purified by HPLC on a preparative C-18 reverse phase silica gelcolumn using a gradient of 70-100% methanol in water. Compound 8 wascollected in the peak of retention time of 13.5 minutes. Proton NMR δ1.72 (m, CH₂CH ₂CH₂NHCH₂), 1.95 (m, CH ₂CH₂CH₂NHCH₂), 2.46 (s, CH ₃Ar),ca. 3.34 (s, —N⁺(CH ₃)₂—), 3.4-4.1 (several m, 9H, carbohydrate C3-C6protons and CH ₂NCH ₂), 6.70 (d, 2 aryl H ortho to NH), 7.28 (d,benzothiazole C5—H), 7.71 (s, benzothiazole C7—H), 7.76 (d,benzothiazole C4—H), 7.80 (d, 2 aryl H meta to NH).

EXAMPLE 4 A Thioflavin-T Amadori Product Binds Amyloid

The present Example demonstrates that a modified Thioflavin-T, to whichan Amadori product has been conjugated, co-precipitates with β-amyloidin vitro. This observation demonstrates the feasibility of linking anAmadori product or AGE to Thioflavin-T for targeting to amyloiddeposits.

A preparation of Compound 8, supra, was employed in this assay. Solubleβ-amyloid peptide was allowed to aggregate in the presence of phosphatebuffer, Compound 8 or dithionitrobenzene and various compounds asdescribed in Example 1, supra. The samples containing dithionitrobenzeneare controls for non-specific association of a chromophoric smallmolecule with precipitated β-amyloid peptide. An additional controlsample was prepared containing phosphate buffer and the variouscompounds, but lacking β-amyloid peptide. After overnight incubation,the tubes were centrifuged at 15,000×g for 30 minutes, half of thesupernatant removed and added to a cuvette, and the absorbance spectrumof the supernatant from 200 to 600 nm obtained. In the absence of anassociation between compound 8 and aggregated sedimentable B-amyloidcomponents of the aggregated assay incubation, compound 8, which has anabsorbance maximum at about 350 nm, will remain in the supernatant andits presence and concentration can be readily determined. Alternatively,association of Compound 8 with the precipitated β-amyloid peptide wouldresult in decreased absorbance at 350 nm. Thus, a decrease in theabsorbance at 350 nm indicates that Compound 8 binds to fibrillarβ-amyloid that sediments during centrifugation under these conditions,as shown in Example 1, supra. In the control tubes, no decrease in theabsorbance is expected, since the compound should remain soluble.

FIG. 7A shows that the absorbance at 350 nm of a supernatant solutioncontaining Compound 8 is reduced when fibrillar β-amyloid peptidepresent in the solution was pelleted, compared to the absorbance priorto pelleting (FIG. 7B). In contrast, the absorbance ofdithionitrobenzene, which has not been reported to bind to aggregatedβ-amyloid peptide deposits, does not decrease in the β-amyloid peptidesample supernatant compared to the control (compare FIG. 7C—pelletformation—with 7D—no pellet formation). These data indicate thatcompound 8 specifically associates with insoluble, aggregated β-amyloidpeptide deposits.

EXAMPLE 5 Age-Amyloid in Type Ii Diabetes

Type II diabetes is characterized by deposits of aggregated amylinpeptide in the pancreas. After a concentration-dependent lag periodduring in vitro incubations, soluble preparations of synthetic amylinslowly form fibrillar aggregates that resemble natural amyloid (Lorenzoet al., 1994, Nature 368:756-760) and are measurable by electronmicroscopy or by Congo Red birefringence under polarized light (Fraseret al., 1991, Biophys. J. 90:1194-1201). Aggregation of soluble amylinin these in vitro assays is expected to be enhanced by addition of smallamounts of pre-aggregated amylin “seed” material. These seeds have alsobeen prepared herein using a naturally occurring reaction betweenglucose and protein amino groups resulting in the formation of advancedglycosylation endproducts (AGEs) which chemically crosslink proteins.AGE-modified amylin-nucleation seeds are expected to further accelerateaggregation of soluble amylin compared to non-modified “seed” material.Over time, nonenzymatic advanced glycosylation, which is likely to occurat lysine-1 of amylin, and may occur at arginine-10, also results in thegradual accumulation of a set of post-translational covalent adducts onlong-lived proteins in vivo. Using a standardized competitive ELISAassay, plaque fractions of Type II pancreatic islet cells are expectedto be found to contain more AGE adducts per mg protein than found inlike preparations from healthy, age-matched controls. These resultsindicate that the in vivo half-life of amylin is prolonged in Type IIdiabetes, resulting in greater accumulation of AGE modifications, whichin turn can act to promote accumulation of additional amyloid.

Materials and Methods

Aggregation and Seeding Reactions.

Synthetic, HPLC-purified peptides representing the 37 amino acid humanor cat islet amyloid polypeptide or amylin may be obtained by synthesisor from a commercial source, such as Bachem (Torrance, Calif.) orPeninsula Laboratories. Quantitative aggregate formation withsub-millimolar amylin concentrations may be detected using the procedureof LeVine (1992, Protein Science 2:404-410). Briefly, fluorescence ofaggregates added to 10 μM Thioflavin-T (Aldrich)/50 mM potassiumphosphate buffer, pH 6.0, can be measured upon excitation at 450±5 nm,and detection of emission at 482±10 nm on a Perkin Elmer LS-50Bspectrofluorimeter. Alternatively, Congo red birefringence underpolarized light can be used to detect aggregation (Fraser et al.,supra). Small amounts of pre-formed aggregates or “nucleation seeds” areadded to the soluble amylin and aggregation initiated, e.g., with 0.1 Msodium acetate.

Generations of “Seeds”.

Soluble amylin (250 /AM) and 0.2 M sodium phosphate buffer, pH 7.5, areincubated with or without 1 M glucose at 37° C. to generate pre-formedaggregates of AGE-amylin referred to as “AGE-amylin seed” or “amylinseed,” respectively. After incubation, protein concentrations of seedpreparations are measured and adjusted with buffer to 150 /AM finalconcentration. Using competitive ELISA (Makita et al., supra), AGEcontent of an AGE-amylin seed and amylin-seed can be determined. Glucoseand/or aminoguanidine, a potent inhibitor of advanced glycation andcrosslink formation (Brownlee et al, 1986, Science 232:1629-1632), arealso added to solutions of amylin before sodium acetate in someexperiments to evaluate whether these AGE inhibitors inhibit seedformation.

Aggregation of amylin at lower (physiological) concentrations may bequantitated by the method of Burdick et al. (1992, J. Biol. Chem.267:546-554). Synthetic preparations of amylin are labeled with ¹²⁵I(NEN) and chloramine-T (Sigma) for 1 minute before the reaction wasquenched with 10 mM tyrosine and sodium meta-bisulfite. Unincorporatedlabel is removed by filtration through a SEPHADEX G-10 columnequilibrated in 0.5×phosphate buffered saline (PBS), pH 7.4. The¹²⁵1-labeled amylin is immediately diluted to 5 nM final concentrationin the presence of various “seeds,” glucose and/or aminoguanidine at theindicated concentrations. After various incubation periods at 37° C.,aggregation reactions are underlayed with 20% sucrose/0.1 M sodiumacetate at the same pH as the incubation mixture, centrifuged for 30minutes at 50,000×g, and frozen in liquid nitrogen. Each microfuge tubeis cut and the bottom 5 mm representing the aggregated sedimentablefraction which have pelleted through the sucrose cushion, is counted ina gamma counter. The remainder of the tube and liquid are also counted.The amount of aggregate formed is calculated as a percentage equal tothe number of counts in the pellet divided by the total number of countsper tube (pellet+remainder) multiplied by 100.

Measurement of AGEs with Competitive ELISA.

Aliquots of pancreas containing amyloid fibrils from patients with andwithout Type II diabetes are resuspended in 2% sodium dodecylsulfate(SDS)/0.1 M β-mercaptoethanol (ME), and Dounce homogenized. Thehomogenate is boiled for 10 minutes and then centrifuged at 10,000×g for10 minutes. Supernatants are aspirated and the resulting pellets washedthree times with PBS at 10,000×g for 10 minutes. This crude plaquefraction may be further washed twice with 4 M urea and twice more withPBS before protease digestion. PBS-washed pellets are resuspended in PBSand 0.1% Proteinase-K (Boehringer Mannheim), digested overnight at 37°C. and heat inactivated at 75° C. for 3 hours. Quadruplicate aliquots ofdifferent amounts of plaque-containing pellet fractions are assayed forAGE content using a competitive ELISA (Makita et al., 1992, J. Biol.Chem. 267:5133-38), against standardized preparation of AGE-modifiedbovine serum albumin (AGE-BSA). Only values in the linear range of thestandard curve should be included in the analyses.

Alternatively, tissue samples or amyloid fibril extracts can be preparedas described (Westermark et al., 1986, Biochem. Biophys. Res. Commun.140:827-831; Westermark et al., 1987, Proc. Natl. Acad. Sci. USA84:3881-85; Westermark et al., 1987, Am. J. Physiol. 127:414-417; Cooperet al., 1987, Proc. Natl. Acad. Sci. USA 84:8628-32).

Protein amounts may be quantitated with micro-BCA kit (Pierce) and withfluorescamine (Bohlen et al., 1973, Arch. Biochem. Biophys.155:213-220). AGE Units are interpolated from a standard dilution curveof AGE-BSA and divided by the sample protein concentration to give AGEUnits per mg protein. Statistical analysis using Student's test can beperformed with the StatWorks program for Macintosh using a Macintoshpersonal computer (Apple Computer, Inc.).

Results

This experiment is expected to show that an AGE-amyloid seed comprisingAGE-modified amylin will increase the rate of amylin aggregation.Similarly, soluble AGE-amylin is expected to aggregate more readily thansoluble amylin. These results will indicate that, as discussed inExample 1, supra, with respect to βAP aggregation in Alzheimer'sdisease, AGE-modification of amylin plays a role in pathogenicamyloidosis associated with Type II diabetes.

This invention may be embodied in other forms or carried out in otherways without departing from the spirit or essential characteristicsthereof. The present disclosure is therefore to be considered as in allrespects illustrative and not restrictive, the scope of the inventionbeing indicated by the appended claims, and all changes which comewithin the meaning and range of equivalency are intended to be embracedtherein.

Various references are cited throughout this specification, each ofwhich is incorporated herein by reference in its entirety.

What is claimed is:
 1. A method for inhibiting AGE-amyloid-dependentaggregation of amyloid peptide and consequent amyloidogenesis in mammalcomprising administering to sand mammal an effective amyloid aggregatinginhibiting amount of an AGE-amyloid formation-inhibiting agent selectedfrom the group consisting of aminoguanidine, α-hydrazinohistidine,lysine, an analog of aminoguanidine, a pharmaceutically acceptable acidaddition salt of al analog of aminoguanidine, and mixtures thereof, saidanalog selected from hydrazine derivatives of the formula:

wherein R is a group of the formula

and R₁ is hydrogen or a lower alkyl group of 1-6 carbon atoms, ahydroxyethyl group, or together with R₂ may be a lower alkylene bridgeof 24 carbon atoms; R₂ is hydrogen or a lower group alkyl of 1-6 carbonatoms or together with R₁ or R₃ is a lower alkylene bridge of 2-4 carbonatoms, amino, hydroxy, or an aminoalkylene group of the formula

wherein n is an integer of 2-7 and R₆ and R₇ are independently a loweralkyl group of 1-6 carbon atoms or together form a part of a cycloalkylor heterocyclic ring containing from 1 to 2 heteroatoms, of which atleast one is nitrogen; and the second of said heteroatoms is selectedfrom the group consisting of nitrogen, oxygen, and sulfur, with theproviso that when the second of said heteroatoms of the heterocyclicring is nitrogen and forms a piperazine ring; said second heteroatom maybe optionally substituted by a substituent that is identical to theportion of the compound on the first nitrogen of the piperazine ring; R₃is hydrogen, a lower alkyl group of 1-6 carbon atoms, or together withR₂ or R₄ is a lower alkylene bridge of 2-4 carbon atoms; R₄ is hydrogen,a lower alkyl group of 1-6 carbon atoms or together with R₃ is a loweralkylene bridge of 2-4 carbon atoms; or an amino group; R₅ is hydrogen,or a lower alkyl group of 1-6 carbon atoms; with the proviso that atleast one of R₁, R₂, R₃, R₄ or R₅ is other than hydrogen; or R is anacyl or a lower alkylsulfonyl group of up to 10 carbon atoms and R₁ ishydrogen.
 2. The method of claim 1, wherein said compound isadministered intraventricularly, intracranially, intraperitoneally,intravenously, intra-arterially, nasally, or orally.
 3. The method ofclaim 1 wherein said amyloid peptide is selected from the groupconsisting of amylin, β-amyloid peptide, and prion protein.
 4. A methodfor inhibiting AGE-amyloid-dependent aggregation of amyloid peptide andconsequent amyloidogenesis in a mammal comprising administering to saidmammal an effective amyloid aggregating inhibiting amount of anAGE-amyloid formation-inhibiting agent selected from the groupconsisting of aminoguanidine, α-hydrazinohistidine, lysine, an analog ofaminoguanidine, a pharmaceutically acceptable acid addition salt of ananalog of aminoguanidine, and mixtures thereof.
 5. The method of claim 4wherein said analog of aminoguanidine is a hydrazine derivative of theformula:

wherein R is a group of the formula

and R₁ is hydrogen or a lower alkyl group of 1-6 carbon atoms, ahydroxyethyl group, or together with R₂ may be a lower alkylene bridgeof 2-4 carbon atoms; R₂ is hydrogen or a lower group alkyl of 1-6 carbonatoms or together with R₁ or R₃ is a lower alkylene bridge of 2-4 carbonatoms, amino, hydroxy, or an aminoalkylene group of the formula

wherein n is an integer of 2-7 and R₆ and R₇ are independently a loweralkyl group of 1-6 carbon atoms or together form a part of a cycloalkylor heterocyclic ring containing from 1 to 2 heteroatoms, of which atleast one is nitrogen; and the second of said heteroatoms is selectedfrom the group consisting of nitrogen, oxygen, and sulfur; with theproviso that when the second of said heteroatoms of the heterocyclicring is nitrogen and forms a piperazine ring; said second heteroatom maybe optionally substituted by a substituent that is identical to theportion of the compound on the first nitrogen of the piperazine ring; R₃is hydrogen, a lower alkyl group of 1-6 carbon atoms, or together withR₂ or R₄ is a lower alkylene bridge of 2-4 carbon atoms; R₄ is hydrogen,a lower alkyl group of 1-6 carbon atoms or together with R₃ is a loweralkylene bridge of 2-4 carbon atoms; or an amino group; R₅ is hydrogen,or a lower alkyl group of 1-6 carbon atoms; with the proviso that atleast one of R₁, R₂, R₃, R₄ or R₅ is other than hydrogen; or R is anacyl or a lower alkylsulfonyl group of up to 10 carbon atoms and R₁ ishydrogen.